B Y T TRNG I HC DC H NI L TH LOAN NH GI TC DNG IU TR BNG CA EB12TRN THC NGHIM H NI-2013 KHA LUN TT NGHIP DC S Ngi hng dn: 1.Ths. Nguyn Thu Hng 2.Ts. L Minh H Ni thc hin: B mn Dc Lc LI CM N Vi lng knh trng v bit n su sc, em xin gi li cm n chn thnh ti Ths. Nguyn Thu Hng, Ts. L Minh H v Ths. Th Nguyt Qu - nhng ngi thy tn tnh hng dn em trong sut qu trnh thc nghim, truyn t nhiu kinh nghim v kin thc chuyn mn qu bu gip em hon thnh kha lun ny. Em xin gi li cm n chn thnh n cc thy c gio, cc anh ch k thut vin b mn Dc lc, b mn Vi sinh-Sinh vt, b mn Bo ch- Cng nghip dc nhit tnh gip , to mi iu kin thun li cho em trong thi gian lm thc nghim ti b mn. Em cng xin gi li cm n ti ton th cn b, ging vin trng i Hc Dc H Ni v du dt, dy bo tn tnh trong sut thi gian em hc tp ti y. Cui cng, em xin by t lng bit n su sc ti cha m, ngi thn v bn b - nhng ngi lun bn cnh ng vin, yu qu v chm lo cho em trong cuc sng. MC LC Trang DANH MC CC CH VIT TT.................................................................... 5 DANH MC CC BNG..................................................................................... 6 DANH MC CC HNH...................................................................................... 7 T VN ........................................................................................................ 1 Chng 1: TNG QUAN ..................................................................................... 3 1.1.Tng quan v bng ....................................................................................... 3 1.1.1.nh ngha v dch t bng..........................................................................3 1.1.2.Mc nng ca tn thng bng .............................................................. 3 1.1.3.Din bin ca tn thng bng....................................................................7 1.1.4.Hu qu ca bng...................................................................................... 12 1.1.5.Cc thuc iu tr ti ch tn thng bng ................................................ 12 1.2.Tng quan v m hnh nghin cu bng ................................................... 14 1.2.1.Cc phng php gy bng nhit thc nghim.......................................... 14 1.2.2.ng vt thc nghim............................................................................... 16 1.2.3.Ch tiu nh gi ....................................................................................... 17 1.3.Tng quan v cht liu nghin cu............................................................ 19 1.3.1. Ngun gc, cu trc ha hc, tnh cht ........................................................ 19 1.3.2. Tc dng...................................................................................................... 20 Chng 2: I TNG V PHNG PHP NGHIN CU...................... 22 2.1. Nguyn liu, ha cht, thit b nghin cu................................................... 22 2.1.1. Mu nghin cu........................................................................................... 22 2.1.2. Ha cht, thuc th...................................................................................... 22 2.1.3. Thit b, my mc phc v nghin cu ........................................................ 23 2.1.4. ng vt, vi sinh vt nghin cu.................................................................. 23 2.2. Ni dung nghin cu..................................................................................... 24 2.3. Phng php nghin cu ............................................................................. 24 2.3.1. Nghin cu hot tnh khng khun, khng nm in vitro ca EB12 ............... 24 2.3.2. Nghin cu tc dng iu tr ti ch ca EB12 trn bng nhit thc nghim............................................................................................................... 25 2.4. Phng php x l s liu ............................................................................ 28 Chng 3: KT QU NGHIN CU............................................................... 29 3.1. Trin khai m hnh gy bng nhit thc nghim........................................ 29 3.2. Tc dng khng khun, khng nm in vitro ca EB12............................... 31 3.3. Tc dng iu tr ti ch ca EB12 trn bng nhit thc nghim.............. 32 3.3.1. Tnh trng chung ca th ............................................................................. 32 3.3.2. Din bin i th ti vt bng....................................................................... 32 3.3.3. Din bin cu trc vi th ti vt bng........................................................... 37 3.3.4. Din bin ca qun th vi khun ti vt bng............................................... 38 Chng 4: BN LUN....................................................................................... 40 4.1.V m hnh nghin cu................................................................................ 40 4.1.1. La chn m hnh nghin cu...................................................................... 40 4.1.2. La chn thi gian gy bng........................................................................ 40 4.2. V tc dng khng khun, khng nm in vitro ca EB12.. ..................... 41 4.3. V tc dng iu tr ti ch ca EB12 trn bng nhit thc nghim.......... 43 4.3.1. Tc dng khng khun khi iu tr ti ch vt bng ..................................... 43 4.3.2. Tc dng chng vim, lm sch vt bng..................................................... 46 4.3.3. Tc dng trn qu trnh lin vt bng........................................................... 48 KT LUN.......................................................................................................... 50 KIN NGH......................................................................................................... 50 DANH MC CC CH VIT TT Aci. Lwofii: Acinetobacter lwoffii AND: acid 2-deoxyribonucleic ARNm: acid Ribonucleic messenger E. american: Eleutherin american E. bulbosa: Eleutherin bulbosa E. cloacea: Enterobacter cloacea E.coli: Escherichia coli EGF: epidermal growth factor E. indica: Eleutherin indica Ent. Faecium: Enterococcus faecium FGF: fibroblast growth factor GAG: glycosaminoglycan HE: hematoxylin-eosin K. pneumoniae: KlEB12siella pneumoniae PDGF: platelet derived growth factor Pro. Mirabillis: Proteus mirabilis S.aureus: Staphylococcus aureus Str. Agalactiae: Streptoccus agalactiae TGF- : transforming growth factor VEGF: vascular endothelial growth factor VK: vi khun. DANH MC CC BNG Trang Bng 1.1:Phn loi su tn thng bng5 Bng 1.2:Phn loi mc nng ca bng7 Bng 2.1:Thang im nh gi tnh trng i th ti vt bng26 Bng 3.1:ng knh vng v khun ca cc mu th i vi cc loi vi khun 31 Bng 3.2:Kt qu nh gi din bin i th ti vt bng35 Bng 3.3:Mc thu hp din tch vt bng36 Bng 3.4:Thi gian lin vt bng36 Bng 3.5:T l cc vi khun phn lp c ti vt bng38 Bng 3.6S lng vi khun S. aureus trn 1 cm2 din tch vt bng39 DANH MC CC HNH Trang Hnh 1.1:Phn loi su tn thng bng5 Hnh 1.2:M hnh 3 vng trong chn thng bng ca Jackson D.7 Hnh 1.3:Cu to ha hc ca eleutherin v isoeleutherin20 Hnh 2.1:S nghin cu tc dng ca EB12 trn bng nhit thc nghim25 Hnh 3.1:Qu trnh gy bng thc nghim29 Hnh 3.2:Hnh nh vt thng sau khi gy bng29 Hnh 3.3:Hnh nh vi th vt bng vi thi gian gy bng 35 giy30 Hnh 3.4:Hnh nh vi th vt bng vi thi gian gy bng 40 giy30 Hnh 3.5:Hnh nh vi th vt bng vi thi gian gy bng 50 giy31 Hnh 3.6:Hnh nh cc vt bng vo ngy th 0 v ngy th 7 bi mu th33 Hnh 3.7:Hnh nh cc vt bng vo ngy th 14 v 21 bi mu th34 Hnh 3.8:Hnh nh m ht ti to, c vim mn tnh37 Hnh 3.9:Hnh nh tn thng bng ang ti to37 1 T VN Bng l chn thng thng gp trong lao ng sn xut cng nh sinh hot thng ngy. nc ta, bng ng hng th 3 trong s cc chn thng ngoi khoa ni chung [13] vi 844.000 bnh nhn mi nm, chim gn 1% dn s [12]. Bng c th do nhiu nguyn nhn gy ra, trong bng nhit thng gp nht chim khong 84-94% s ca bng [17]. Bnh nhn bng thng phi iu tr di ngy, tn km. Nu khng c iu tr tt, bng c th li nhiu di chng lu di, nh hng ti sinh hot hng ngy, kh nng lao ng, thm m v tm l ca ngi bnh [17], [23]. iu tr ti ch c vai tr quan trng i vi qu trnh iu tr bng. Mc ch iu tr ti ch l loi b m hoi t, ngn nga nhim khun, y nhanh qu trnh lin vt thng, hn ch di chng. C nhiu nhm thuc c s dng iu tr ti ch vt thng bng nh: thuc khng khun, thuc lm rng hoi t, thuc lm kh se vt bng, thuc kch thch biu m ha, [17]. Tuy nhin, ngay c nhng thuc c s dng ph bin nht vn c rt nhiu tc dng khng mong mun, thm ch lm chm qu trnh lin vt thng. Chnh v vy, vic tm kim cc thuc mi tt hn nhm h tr hoc thay th cc thuc hin ti lun l yu cu bc thit i vi cng tc iu tr bng hin nay. Sm i hnh l dc liu ph bin, mc hoang v c trng nhiu ni nc ta lm thuc. T lu, Sm i hnh c s dng rng ri trong dn gian cha cc bnh ngoi da nh vim da, mn nht, chc u, l lot [4], [9]. Nghin cu ca mt s tc gi trong v ngoi nc cho thy cc hot cht chnh trong Sm i hnh l eleutherin v isoeleutherin c tc dng khng khun tt i vi mt s vi khun [5], [42]. T ngun dc liu r tin, sn c, vi quy trnh n gin, vin Ha hc cc hp cht thin nhin, vin Khoa hc quc gia Vit Nam chit xut v phn lp thnh cng hn hp 2 hot cht eleutherin v isoeleutherin vi t l 1:1 (gi l EB12) t thn r Sm i hnh. 2 Vi mong mun tm c loi thuc mi t ngun nguyn liu sn c trong nc, da vo cc ti liu tham kho v cc nghin cu thm d, chng ti tin hnh nghin cu ti: nh gi tc dng iu tr bng ca EB12 trn thc nghim vi 2 mc tiu sau: 1. Nghin cu tc dng c ch vi khun, vi nm in vitro ca EB12. 2. NghincutcdngiutrtichcakemEB12trnbngnhitthc nghim.3 Chng 1: TNG QUAN 1.1.TNG QUAN V BNG 1.1.1.nh ngha v dch t bng Bng l cc tn thng do nhit , ha cht hoc dng in gy ra. Tn thng bng thng ch da, nhng cng c trng hp bng su ti cc lp di da nh gn, c, xng khp v cc tng.Bng l chn thng ngoi khoa thng gp. Theo thng k, nc Anh c khong 250.000 bnh nhn bng mi nm [26]. Con s ny M ln n hn 1 triu ngi [48]. Vit Nam c tnh mi nm c khong 844.000 ngi b bng, chim gn 1% dn s, trong 25% l tr em t 1 n 5 tui [12]. Tai nn bng l mt trong nhng gnh nng i vi ngnh y t cc nc. Hng nm, s bnh nhn bng cn iu tr ni tr Php khong 10.000 ngi [17], Hoa K khong 75.000 ngi [48], Anh khong 112.000 ngi [26]. Ti Vit Nam, ch tnh ring Vin Bng Quc Gia mi nm tip nhn v iu tr cho hn 2.500 bnh nhn bng mc t nng n rt nng [17]. Bng li nhng gnh nng thng tt nng n, ng thi l nguyn nhn gy t vong ng lo ngi. Thng k ca t chc Y t Th gii nm 2004 cho thy mi nm c hn 310.000 bnh nhn t vong do bng la, cha k n cc loi bng khc, trong hn 30% bnh nhn di 20 tui [23]. Ti Vit Nam, nghin cu Gnh nng bnh tt v chn thng Vit Nam 2008 ca Nguyn Th Trang Nhung cho thy t l t vong do bng l 243/100.000 dn, cao hn rt nhiu so vi t l chung ca khu vc v th gii [13]. Cng theo nghin cu ny, s nm sng tn tt hiu chnh (DALYs) m tai nn bng li ln ti 15.717/100.000 dn [13]. 1.1.2.Mc nng ca tn thng bng i vi tn thng bng, phn ng ca c th, din bin ca qu trnh lin vt thng, nguy c xut hin cc bin chng v hu qu do bng li ty thuc vo mc nng ca tn thng. nh gi mc nng ca tn thng bng, c th da vo cc yu t sau: su ca tn thng, din tch vt bng, tc nhn gy 4 bng, v tr bng trn c th v th trng bnh nhn [21]. C th: 1.1.2.1. Tc nhn gy bng Cc tc nhn gy bng c th chia thnh ba nhm chnh l nhit , ha cht v dng in [23], [26]. Mc nng ca tn thng ph thuc vo loi tc nhn v c im ca tc nhn gy bng. Cc tc nhn nhit nh ngn la, cc vt rn nng, cc cht lng nng hoc kh nng gy bng nhit. Mc tn thng trong bng nhit ph thuc nhit ca tc nhn, thi gian, din tch v p lc ca ngun nhit ln da [26]. Nhit cng cao, thi gian tip xc cng di, din tch cng rng v p lc ln da cng ln, mc tn thng cng nng [26]. Cc ha cht nh acid hay kim mnh c th gy bng ha cht vi mc nng ca tn thng ph thuc vo bn cht v nng ca tc nhn [21]. Cc acid gy bin tnh protein, hoi t m v thng gy au cho bnh nhn [26]. Ring acid hydrofluoric c kh nng thm nhp su vo m c th, gy nhim c ton thn, c th dn n t vong ngay c vi vt bng nh [26]. Cc tc nhn kim c kh nng thm nhp vo m su hn hu ht cc acid, gy bin tnh protein, lm t bo mt nc v gy hoi t m m [26]. c bit, bng kim ban u khng au nn bnh nhn thng khng bit x tr kp thi khin tn thng nng thm [26]. Mc nng ca tn thng trong bng in ty thuc vo tnh cht v in th ca ngun in. Trong khi cc ngun in mt chiu vi in th thp thng ch gy tn thng ngoi da, ngun in cao th (khong 1000V) c th gy tn thng m rng ln, thm ch tiu c vn v suy thn [26], [49]. Dng in xoay chiu nu i qua c tim c th gy ri lon nhp tim v dn ti t vong [26], [49]. 1.1.2.2. Din tch tn thng bng Din tch vt bng l mt trong cc tiu ch nh gi mc nng ca tn thng: din tch cng rng, mc tn thng cng nng [23]. Vt bng c din tch rng c nguy c nhim khun vt bng, nhim khun ton thn cao [23], [29], [47], thi gian lin vt thng ko di, li so xu [23]. Tng din tch bng trn c th qu ln cn c th dn n sc tun hon v t vong [23]. Thng k cc 5 nc thu nhp thp cho thy nguy c t vong do bng trn 30% tng din tch b mt c th l 50%, i vi bng trn 50% din tch b mt c th l gn 100% [23]. 1.1.2.3. su tn thng bng su tn thng bng quyt nh nh hng ca vt bng n tnh trng ton thn, nguy c nhim khun, kh nng t lin vt thng, thi gian lin vt thng v hu qu sau bng [17], [18]. Da trn su tn thng, c th phn loi bng nh sau: Bng 1.1: Phn loi su tn thng bng [17], [18]. Bng nngBng su III I II III nng III su IV V Vim cp da do bng Tn thng biu b, lp y cn Tn thng lp nh, phn ph ca da cn Tn thng lp li, ch cn phn su tuyn m hi Bng ton b lp da Bng da v cc lp di da, ni tng Hnh 1.1: Phn loi su tn thng bng [61] Bng I: tn thng lp nng ca biu b, cc phn khc nguyn vn, t khi sau 2-3 ngy, khng li so [17], [21]. 6 Bng II: tn thng biu b, phn y cn nguyn vn, t khi sau 7-10 ngy nh biu m t phn cn li ca t bo mm y biu b, li nn nht mu hn mu da lnh xung quanh [17]. Bng III nng: tn thng ti lp nh, cn ng, gc lng, tuyn m hi, tuyn b nhn. T khi sau 12-15 ngy nh biu m ha t cc phn ph cn li ca da [17]. Bng III su: tn thng ti lp li trung b, ch cn phn su ca tuyn m hi. Hnh thnh o biu m t phn cn li ca tuyn m hi. Bng III su rt d chuyn thnh bng su [17]. Bng IV: tn thng su ht lp da, tt c cc thnh phn ca biu m u b ph hy, khng c kh nng t lin do khng c cc thnh phn ca biu m [17]. Bng V: tn thng qua da ti lp c, gn, xng, ni tng, thng li hu qu nng n [17]. 1.1.2.4. V tr bng trn c th V tr bng trn c th cng nh hng n mc nng ca tn thng. Nhng vt bng c cng din tch v su tn thng nhng v tr khc nhau th mc nng khc nhau [21], [29]. Bng vng u mt gy ri lon vn mch dn n thiu mu no, ph no. Bng vng hu mn, sinh dc, nch d b nhim khun. Bng bn tay li so co rt, nh hng ti kh nng vn ng nn c tin lng nng hn. 1.1.2.5. Th trng bnh nhn Bng nhng i tng bnh nhn c bit nh ngi cao tui, tr em, bnh nhn suy gim min dch, bnh nhn ang mc bnh mn tnh, bnh nhn c cc tn thng kt hp c nh gi mc nng hn so vi nhng bnh nhn thng thng [21]. Da trn cc yu t trn, c th phn loi mc nng tn thng bng ca bnh nhn nh sau: 7 Bng 1.2: Phn loi mc nng ca bng [61] Mc i tng Bng mc nh -Bng I. -Bng II c din tch nh hn 15% din tch c th vi ngi ln v nh hn 10% vi tr em. -Bng III vi din tch nh hn 2%. Bng mc trung bnh -Bng II c din tch t 15-25% ngi ln, t 10-20% tr em. -Bng III c din tch t 2-10%. Bng mc nng -Bng II trn 25% ngi ln, trn 20% tr em. -Bng III trn 10%. -Bng in. -Bng cc v tr u, mt, c, tay, chn, b phn sinh dc. -Bng c kt hp gy xng hoc chn thng ng h hp -Bng i tng bnh nhn c bit. 1.1.3.Din bin ca tn thng bng Hnh nh m hc ca tn thng bng c Jackson D. m t gm 3 vng: vng hoi t, vng ph v vng xung huyt [43]. Vng chu tc ng trc tip ca tc nhn gy bng b hoi t m t bo. Vng k cn b ph , phn ng vim xy ra mnh m, cc t bo b tn thng. Vng ngoi cng l vng xung huyt vi hin tng gin mch, cc t bo t b tn thng. Hnh 1.2: M hnh 3 vng trong tn thng bng ca Jackson D. [43]. 8 (1: vng hoi t; 2: vng ph ; 3: vng xung huyt) 1.1.3.1. Cc giai on lin vt bng Da l c quan c nhiu chc phn quan trng i vi c th. y l hng ro bo v cc c quan bn trong trc cc yu t vt l, ha hc v vi sinh vt bn ngoi. Da cn l c quan cm gic, min dch, bi tit, iu ha nhit . Tnh ton vn v cu trc v chc nng ca da c ngha quan trng i vi c th. V vy khi da b tn thng, c th s phn ng li bng cc qu trnh sinh l thit lp li trng thi ban u ca da, cc qu trnh ny gi l qu trnh lin vt thng da. Lin vt thng da l qu trnh sinh l phc tp, gm nhiu giai on, din ra theo trnh t nht nh v chng gi ln nhau. C th phn qu trnh lin vt thng da lm ba giai on chnh: (1) giai on vim cp, (2) giai on tng sinh, (3) giai on sa cha vt thng v hnh thnh so [1], [17], [18], [28]. Giai on vim cp Mc ch ca giai on ny l ngn nga s lan rng ca cc tc nhn gy hi n cc m ln cn, loi b cc mm bnh v cc mnh vn t bo, to c s cho qu trnh phc hi [1], [17], [28]. Phn ng vim xut hin ngay khi da b tn thng, vi biu hin sm nht l ri lon tun hon ti ch. Ri lon tun hon ti v tr vim c c trng bi cc hin tng: ri lon vn mch, to dch r vim, bch cu xuyn mch v hin tng thc bo [1]. Ri lon vn mch l chui cc phn ng lin tip bao gm: co mch, sung huyt ng mch, sung huyt tnh mch v mu. Phn x co mch xy ra ngay khi m b tn thng, ko di trong thi gian rt ngn nhng c vai tr quan trng trong vic thc y phn ng dy chuyn tip theo l sung huyt ng mch. Sung huyt ng mch gip vim c ti mu giu oxy v nng lng, to iu kin cho qu trnh thc bo xy ra. Ch khi qu trnh thc bo hon tt hin tng sung huyt ng mch mi yu dn i v chuyn sang sung huyt tnh mch. Trong sung huyt tnh mch, cc mao mch gin rng, cc t bo vng trung tm vim ha vo lp huyt tng bao quanh, vim c dn sch, ngn cn s lan rng ca cc tc 9 nhn gy bnh. Dch r vim c hnh thnh ti v tr vim, vi hai nhm thnh phn c bn l cc thnh phn ca mu thot ra (protein huyt tng, cc cht in gii, hng cu, bch cu, tiu cu) v cc thnh phn mi hnh thnh do ri lon chuyn ha v tn thng m (histamin, serotonin, acetylcholin, bradykinin, leucotrien, cc sn phm chuyn ha ca acid arachidonic, cc cytokin interleukin-1, interleukin -6, interleukin-8, cc cht gy st, cc acid nhn, cc enzym,) [1]. Dch r vim c vai tr bo v, nhng lng nhiu qu c th gy chn p m gy au nhc thm ch hn ch hot ng ca cc c quan xung quanh. Hin tng bch cu xuyn mch l h qu ca s tng tnh thm ca thnh mch do tc dng ca cc cht ha ng ng (cc thnh phn ca VK b dung gii, cc sn phm hot ha ca b th, cc chemokin,..) [1]. Bch cu di chuyn v pha c phn ng vim, bm vo thnh mch, xuyn qua vch mch v tin ti vim lm nhim v thc bo. Mc ch ca thc bo ti vim l dn sch cc t bo c hi, bo v c th. Tuy nhin cc phn ng thc bo ti vim cng c th gy ri lon chuyn ha hoc tn thng m th pht, lm tn thng thm trm trng v gy kh khn cho qu trnh lin vt thng [1]. Giai on tng sinh Cui giai on vim cp, giai on tng sinh hnh thnh m mi bt u. Cc vt bng nng nh bng II c th t lin nh s phn bo ca cc t bo lp y biu b v bit ha thnh cc lp t bo ca biu b [17], [18]. Vi bng su t III tr i, qu trnh ti to m bao gm s hnh thnh m ht v qu trnh biu m ha ph kn m ht bng cc t bo biu m [17], [18]. M ht bao gm cc t bo mi v cc mch mu mi. Cc mch mu mi c hnh thnh t cc t bo ni m tnh mch nh cn st li vng tn thng, di tc dng kch thch ca cc yu t tng trng: EGF, PDGF, TGF-, FGF, VEGF. Trong khi , cc t bo mi c hnh thnh t nguyn bo si. Nguyn bo si vn l cc t bo si trong dch gian bo, di tc dng kch thch ca dch r vim 10 m hot ha thnh nguyn bo si, c kh nng tng hp nhiu loi protein ca m nn nh collagen, fibrinonectin, glycosaminglycan (GAG) [1], [18], [28]. Sau khi m ht hnh thnh, qu trnh biu m ha xy ra ph kn m ht. Qu trnh ny gm ba thi k lin tip nhau: (1) s di c ca cc t bo sng t ra vt thng vo vng tn thng, (2) s phn chia ca cc t bo lp y to ra cc t bo ngun, (3) s trng thnh ca cc t bo biu m tr thnh cc lp t bo y ca biu b [57], [28]. Giai on sa cha vt thng v hnh thnh so L giai on di nht ca qu trnh lin vt thng [28]. M lin kt c to ra v lin tc c sa cha m bo to so da bn chc. Cc si collagen c to ra bi nguyn bo si v b ph hy bi cc collagenase khng ngng ti cu trc collagen. Cc collagen ny cng vi cc si khc ca m lin kt khng ngng c nh hng, sp xp li to ra m so bn chc. Nu c s mt cn bng trong trng thi ti lp collagen s dn n trng thi khng bnh thng ca so: so ph i, so li hoc so teo [28]. 1.1.3.2. Nhim khun vt bng Nhim khun l bin chng hay gp trong qu trnh din bin ca tn thng bng bi vt bng t n to ra nhng iu kin ti ch v ton thn thun li cho s pht trin v lan rng ca vi sinh vt [29], [47]. Ti ch: tn thng bng lm mt tnh ton vn ca da, ph v hng ro bo v t nhin ca c th, to iu kin cho vi sinh vt trn da v t mi trng xm nhp vo vt thng [29], [31]. Ti y, s phn gii protein v lipid ca m tn thng to ra mi trng dinh dng thun li cho s pht trin ca VK [29], [31]. Hn na, di tc dng ca phn ng vim, cc mao mch b bt tc khin cho cc t bo min dch, cc yu t bo v trong dch th c th cng nh khng sinh dng ng ton thn kh c th theo ng tun hon ti m b bng [31]. Ton thn: tn thng bng c th dn n suy gim min dch, gim nng cc khng th dch th, gim hot tnh b th, gim chc nng ca i thc bo, gim s lng bch cu a nhn trung tnh, lympho bo do gim kh nng 11 chng ca c th trc s pht trin ca VK [29], [31]. Ngoi ra, ri lon huyt ng do bng dn n thiu mu nui dng cc c quan, trong c h tiu ha, lm suy yu hng ro bo v nim mc thnh rut, to iu kin cho VK xm nhp vo h tun hon chung [31]. Cc nghin cu cho thy trn b mt ca hu ht cc vt bng u c mt hoc nhiu loi VK [7], [37]. Tuy nhin nguy c nhim khun ph thuc vo s lng v c lc ca VK trn vt bng [29]. Cc loi VK thng gy nhim khun bng l S. aureus, P. aeruginosa v cc trc khun ng rut [7], [20], [29], [31], [47], [48]. S. aureus l nguyn nhn hng u gy nhim khun vt bng, ng thi cng l mt trong nhng nguyn nhn chnh gy nhim khun huyt [29], [37], [16], [7], [2], [20], [50]. y l loi VK nguy him, c nhiu c t, khi vo c th gy nhiu bin chng nng n [29], [31]. Nghin cu ca L Th Kim Anh (1997) v Nguyn Nh Lm (2010) cho thy cc chng S. aureus phn lp c ti vt bng u c tnh khng thuc cao i vi hu ht cc khng sinh thng thng [2], [7]. Hn na, s c mt ca S. aureus ti vt bng cn lm tng tnh khng thuc [7] v tng kh nng xm nhp vo m ca cc VK khc [31]. P. aeruginosa l loi VK c c lc cao, c kh nng xm nhp su vo cc m [20], [29], [37], [47], [50]. y l nguyn nhn chnh gy nhim khun huyt v nhim khun ton thn trong bng [20], [29] vi t l phn lp c trong mu c khi ln ti 62,5% [20]. P. aeruginosa l loi VK nguy him, khng hu ht cc loi khng sinh thng dng vi t l khng cao [7], [29], [47]. Bnh nhn bng nhim khun huyt do P. aeruginosa thng c tin lng xu, t l t vong cao [20]. Tnh trng nhim khun c th thc y phn ng vim qu mc, chuyn bng nng thnh bng su, gy hoi t t chc, thi mnh da ghp [47], [50]. Vi khun t vt bng c th xm nhp su vo c th, gy nhim khun huyt, nhim khun ton thn [37], [29]. Trong iu tr, nhim khun bng l bin chng nguy him, l nguyn nhn hng u gy t vong cho bnh nhn [29], [31], [37], [47], [48], [50]. 12 1.1.4.Hu qu ca bng Bng li hu qu nng n cho bnh nhn, gia nh v x hi. i vi bnh nhn, tn thng bng da, ni tp trung u mt ca cc dy thn kinh cm gic, khin ngi bnh au n, cn dng nhiu bin php h tr gim au [8], [17]. Nhng trng hp bng su, bng din rng, lng huyt tng thot ra ngoi mch qu ln mang theo cc cht in gii, protein c th khin bnh nhn sc bng, suy thn cp, thm ch t vong [8], [17]. Cc bin chng nh nhim c bng, st do hp thu m bng, nhim khun vt bng, nhim khun ton thn cng nh hng n sc khe, tnh mng ca bnh nhn [8], [17], [37]. Cc vt bng cn trn 3 tun lin vt thng thng li cc di chng lu di nh so, co rt da, bin dng khp [49]. So bng khng ch nh hng n thm m, tm l m cn c th hn ch vn ng, nh hng n hot ng thng ngy v kh nng lao ng ca bnh nhn [8], [23], [48]. Vi gia nh v x hi, gnh nng trc tin ca bng l chi ph iu tr. Chi ph iu tr bng ty thuc vo loi bng v mc nghim trng ca bng [23]. Anh, chi ph trung bnh ca mt ca bng nc nh l 1.850 bng Anh (tng ng 3.618 la M), chi ph ny Hoa K vo khong 1.187 la M [23]. Cc vn khc nh phc hi chc nng lu di, tn tt, tht nghip, kh ha nhp cng ng cng l nhng gnh nng m bng li [23]. 1.1.5.Cc thuc iu tr ti ch tn thng bng iu tr ti ch gi vai tr quan trng trong qu trnh iu tr bng. Mc ch iu tr ti ch l nhm nhanh chng loi b m hoi t, c ch spht trin ca VK, kch thch qu trnh biu m ha, hn ch bin chng. Ty vo tc dng, cc thuc iu tr ti ch c th c phn thnh cc nhm nh sau:1.1.5.1. Cc thuc khng khun ti ch vt thng bng Cc thuc khng khun ti ch vt thng bng thng dng: -Cc dung dch st khun: povidon- iodin (betadin 10%), nitrat bc 0,5% [29]. 13 -Cckhngsinhhochnhpcckhngsinh:kemsulfadiazin-bc1%, Mafenide acetat (Sulfamylon) dng kem 10% hoc dung dch 5%, kem gentamycin 0,1%, thuc m polymycin,[29], [19], [37], [55]. -Cc thuc y hc c truyn: m Maduxin ch t l sn v du sn [14]. Trong s cc thuc iu tr ti ch vt bng, Kem sulfadiazin-bc 1% (SSD) l thuc c s dng rng ri nc ta v nhiu nc trn th gii do c ph khng khun rng, tc dng ln hu ht cc VK v mt s vi nm gy bnh [16], [18], [19], [29], [37], [48], [49]. Tc dng ca SSD ch yu do tc dng dit khun ca bc phi hp vi tc dng km khun ca sulfadiazin [29], [55]. Tuy nhin, thuc c mt s nhc im sau: -Lm chm tiu hy v rng hoi t [52], lm mt hot tnh ca cc enzym lm rng hoi t khi dng kt hp [3]. -Lm chm lnh vt thng [15], [44]. -Sulfadiazin c hp thu vo c th v gy nhng tc dng khng mong mun nh: gim bch cu, tn thng thn, tng men gan, thiu mu tan huyt, c bit l nhng trng hp bng din rng [3], [15], [45]. Do vic dng thuc ph bin v ko di, SSD b mt s vi khun khng li, c bit l S. aureus, P.aeruginosa, Klesiella[16], [18], [27], [37], [52].1.1.5.2. Cc thuc lm rng hoi t vt bng S dng cc thuc lm rng hoi t vt bng nhm loi tr nhanh m hoi t, loi b mi trng thun li cho VK pht trin, to iu kin cho qu trnh lin vt thng xy ra. Cc thuc thng dng bao gm: cc enzym tiu hy protein c ngun gc ng vt, thc vt hoc vi sinh vt; N-acetylcystein; cc acid yu nh acid salicylic,[18]. 1.1.5.3. Cc thuc kch thch biu m ha v to m ht Cc thuc trong nhm ny ch yu c ngun gc ng vt v thc vt nh: m Maduxin (t du sn v l sn), m Madecasol (t cy rau m), kem ngh, du gc, du gan c,[18]. 1.1.5.4. Cc thuc lm kh se vt bng 14 Thnh phn ca cc thuc lm kh se vt bng thng cha tanin, c tc dng lm ng dch vt thng, kt ta protein, lin kt cc t collagen to thnh mt mng [18]. Mt s thuc trong nhm: cao c l sim, cao kho nhm, cao kho vng, cao l trm, ch dy, v xoan tr,[18]. 1.1.5.5. Cc vt liu sinh hc thay th da hoc che ph tm thi S dng cc vt liu che ph nhm mc ch gim au cho bnh nhn, hn ch mt dch, mt mu qua vt thng, hn ch nhim khun vt bng, kch thch biu m ha vt bng nng, kch thch to m ht bng su [49], [55]. Cc vt liu thng dng c th l da ng vt (da ch ti, da ch ng kh, da ln), da ngi (da t thi, da t thn bnh nhn) hoc vt liu c ngun gc sinh tng hp: mng Biobrane, mng Intergra, [49], [55]. 1.2.TNG QUAN V M HNH NGHIN CU BNG Nghin cu tc dng ca thuc trn cc m hnh ng vt l giai on bt buc i vi qu trnh nghin cu v pht trin thuc ni chung cng nh nghin cu v pht trin thuc iu tr bng ni ring. V vy, cc m hnh thc nghim trn ng vt c vai tr rt quan trng. Nhiu m hnh nghin cu v bng c pht trin, xut da trn nguyn tc chung l s dng tc nhn thch hp to tn thng bng trn ng vt sao cho cc tn thng ny c din bin sinh l tng t trn ngi [60]. Yu cu chung ca m hnh thc nghim nghin cu bng l to ra nhng tn thng ng nht, kim sot c su vt bng, thc hin trn ng vt sn c, dng c n gin, d tin hnh. Ni dung ca mt m hnh nghin cu bao gm: phng php to tn thng bng v cc ch tiu nh gi. 1.2.1.Cc phng php gy bng trn thc nghim Tng ng vi cc nhm tc nhn chnh gy bng, cc phng php gy bng thc nghim c th c phn loi theo ba nhm chnh: dng in, dng ha cht v dng nhit [53], [32]. Phng php dng in thng i hi cc ng vt bc cao nh ch thm ch ng vt linh trng to ra tn thng bng c din bin sinh l tng t trn ngi [53]. 15 Phng php s dng ha cht gy bng c nhc im l kh kim sot s khuch tn ca ha cht vo da, do thng kh to ra c cc vt bng ng nht v din tch v su [53]. Phng php gy bng s dng nhit d tin hnh, to ra c nhng tn thng ng nht, c p dng rng ri trong nhiu nghin cu [53]. C nhiu m hnh bng nhit thc nghim c pht trin v xut, trong cc ngun nhit c s dng ch yu l nc si, kim loi nng v ngn la. M hnh s dng nc si gy bng c Mason v Walker m t vo nm 1968 [46]. Trong , ng vt thc nghim c gi c nh trn mt dng c kim loi c khe h vng lng, tng ng vi vng da c loi b lng ca con vt. Khi cho ng vt tip xc vi nc si, vng da khng c bo v tip xc trc tip vi nc si v b bng. Nhit ca nc v thi gian tip xc c iu chnh thu c tn thng c su nh mong mun. Din tch khe h cng c tnh ton vt bng c din tch ph hp. y l m hnh bng nhit thc nghim kinh in, to ra nhng tn thng bng ng nht, c p dng kh rng ri trong cc nghin cu [53], [32]. Cc m hnh gy bng bng kim loi nng c nguyn tc chung l s dng dng c kim loi c lm nng n nhit xc nh, tip xc vi vng da c co lng ca con vt di mt p lc khng i, trong khong thi gian thch hp to tn thng bng [32], [53], [57]. M hnh kinh in s dng dng c gy bng l cc ming kim loi. Do p lc gia dng c kim loi v vng da gy bng do k thut vin quyt nh, kh kim sot, cc vt bng c to ra c th khng ng nht v su tn thng [53]. khc phc nhc im ny, mt s m hnh ci tin s dng dng c gy bng l thanh kim loi. Khi , p lc ln vng da gy bng do trng lng ca thanh kim loi quyt nh nn to ra c nhng tn thng bng ng u hn [53]. Pocidalo J.J (1955) v Hladovec I. (1961) s dng cc ng kim loi c th cha nc nng lm ngun nhit gy bng. Phng php ny c th iu chnh p lc ln vng da gy bng bng cch iu chnh th tch nc trong ng kim loi. Hn na, nc trong ng cng gip gi nhit ng 16 kim loi hng nh hn trong thi gian gy bng. Vit Nam, phng php ny c p dng thnh cng trong cc nghin cu ca Nguyn Th T (1989), V Th Ngc Thanh (2003) v Trn Th L (2011) [ 10], [14], [22]. Ngn la cng c dng lm tc nhn gy bng bng cch cho tip xc vi vng da ng vt trong nhng khong thi gian xc nh. Katacura T v cng s xut s dng mt ngn la ga phn tn nhit lm tc nhn gy bng[31], [32]. Trong m hnh ca Stieritz v Holder, ngn la c to ra bng cch tri ethanol ln vng da ng vt v t to vt bng [31], [32]. Cc m hnh ny i hi dng c phc tp, t c p dng trong cc m hnh nghin cu [31]. 1.2.2.ng vt thc nghim Khi la chn ng vt tin hnh cc nghin cu thc nghim v bng, cn quan tm n c im cu trc da, c im sinh l, kch thc c th, gi thnh v tnh sn c ca ng vt [32], [35], [57], [60]. c im cu trc da l tiu ch quan trng la chn ng vt thc nghim bi v mun to ra c nhng tn thng bng c din bin sinh l tng t trn ngi, da ng vt cn c cu to tng t da ngi. Khng k ng vt linh trng, da ln c cu trc gn vi da ngi nht, sau n chut lang, chut cng trng, th, chut nht trng [32], [35], [53], [57]. Do cc nghin cu v bng su thng i hi tin hnh trn ln hoc chut lang [32], [35]. c im sinh l ca ng vt cng l mt tiu ch cn quan tm. Ln thng i hi iu kin gy m, hi m v chm sc sau bng kh phc tp [32], [53]. Th rt d b nhim khun, do thch hp cho cc nghin cu v nhim khun bng, tuy nhin i hi iu kin v sinh nghim ngt trong qu trnh thc nghim [32], [53]. Chut cng trng c c tnh khng khun rt tt, khng yu cu iu kin th nghim qu kht khe, thch hp cho cc nghin cu trong thi gian ko di [53], tuy nhin s dng cho cc nghin cu v nhim khun bng cn phi cy VK vo vt thng [32].Kch thc c th ng vt nh hng n thao tc thc nghim, ng vt cng ln cng d thao tc. Tuy nhin cc ng vt c kch thc ln thng c gi thnh 17 cao, nh hng n chi ph nghin cu [35], [53], [57], [60]. 1.2.3.Ch tiu nh gi Cc ch tiu nh gi trong mt nghin cu ty thuc vo ni dung v mc ch nghin cu . Tuy nhin da vo din bin sinh l ca bng, c th nh gi tnh trng ti ch ca vt thng bng thng qua hai nhm vn chnh: (1) qu trnh ti to tn thng, (2) mc nhim khun vt bng. 1.2.3.1. nh gi qu trnh ti to tn thng bng C th nh gi qu trnh ti to hi phc vt thng bng thng qua cc ch tiu i th, vi th v ha m. Din bin i th ti vt bng -Cccimchungcavtbng nh mc phn,sunghuyt,titdch, tnh trng lot, hoi t vt thng: dng nh gi din bin ca qu trnh lin vt thng,cbitlphnngvimvtnhtrngnhimkhuntich[30],[57], [60]. -Mc thu hp din tch vt bng: cho thy din bin ca giai on tng sinh vi s hnh thnh m ht v biu m ha [30], [57], [36], [60]. Giai on tng sinh c c trng bi hin tng cc t bo biu m di c t ra vt thng vo trung tm,lng ng,tngsinh, hnhthnh biu m mi do dintchvtthngthu hp dn cho thy giai on tng sinh ang din ra. -Thigianlinvtbng:lthigiancnthithonttqutrnhlinvt thng. Thigianlinvtbngcng ngnchngt qu trnhtito phchivt thng xy ra cng nhanh [30], [57]. - bn c hc ca m mi c to thnh: kh nng chu lc, tnh n hi ca m mi c to thnh [57], [60]. Tiu ch ny gip nh gi kt qu ca qu trnh linvtthng,c bit lgiaionsachavtthngv hnhthnhso[57]. Bnchtcaqutrnhlinvtthnglthitlplistonvnvcutrcv chc nng cho da, v vy nh gi bn c hc ca m mi c to thnh l mt tiu ch cn quan tm. Din bin vi th ti vt bng18 Qu trnh ti to v phc hi tn thng bng c th nh gi da trn din bin vi th ti vt bng thng qua cc ch tiu sau: -S lng, mt ca cc loi bch cu, cc t bo thc bo ti m tn thng: th hin mc vim ti m tn thng [30], [36], [57]. Phn ng vim cng mnh, cc t bo vim tp trung cng nhiu ti v tr vt thng. -Mt v cu trc cc mch mu, cc t bo biu m c tn to [30], [57], [60]. -Ch s phn chia t bo lp y biu b: th hin r rng kh nng ti to v phc hi [36], [57]. Din bin ca cc ch s sinh ha ti m tn thng -nhlnghydroxyprolintimtnthng:nhgimctnghp collagen ti m tn thng, cho bit din bin ca giai on tng sinh v to m ht [30],[36],[57].Collagenlthnhphncutochnhcachtnnngoiboca da, chim khong 75% trng lng kh ca da. Tuy nhin hin nay cha c phng phpnhlngtrctipcollagentimtnthng,dophinhlng collagen thng qua hydroxyprolin, mt acid amin c trng ca collagen [36], [57]. -nh lng hexosamin v acid hexuronic ti m tn thng: cho bit mc tng hpglycosaminoglycanv proteoglycant nguynbosi[36], [57].yl nhng cht nn ngoi bo quan trng, to ma trn collagen c th lin kt v lng ng, to iu kin cho giai on sa cha vt thng v n nh cu trc m mi c tn to [57]. 1.2.3.2. nh gi mc nhim khun ti vt thng Trong iu tr, nhim khun l bin chng nguy him, l nguyn nhn t vong hng u cho bnh nhn [29], [31], [37], [47], [48], [50]. Nhim khun vt bng c th thc y phn ng vim qu mc, chuyn bng nng thnh bng su, gy hoi t t chc, thi mnh da ghp [47], [50]. Vi khun t vt bng c th xm nhp su vo c th, gy nhim khun huyt, nhim khun ton thn [37], [29]. V vy, tnh trng nhim khun ti vt thng l mt trong nhng ch tiu cn theo di trong qu trnh iu tr. 19 Mc v nguy c nhim khun vt bng c th nh gi da trn chng loi v mt VK trn b mt vt bng hoc trong m tn thng [37], [47]. Vt bng c mt VK t hn 105VK/1gm thng t c nguy c nhim khun lan trn [18], [29], [37]. Tuy nhin nu mt VK t 107-109VK/1g m, nguy c nhim khun huyt rt cao [18], [37]. xc nh s lng v chng loi VK ti vt bng, c th s dng cc phng php sau: XcnhslngVKhayvinmctrong1gmtnthnghoctrn 1cm2 din tch vt bng [29], [30], [47]. -Phng php xc nh s lng VK c trong 1g m tn thng: cho bit chnh xc nht mc nhim khun vt bng, tuy nhin kh khn trong ly mu v x l mu bnh phm [29], [47]. -Phng php xc nh s lng VK trn 1cm2 din tch vt bng: n gin, d thc hin [29], [39]. Vi bng nng, phng php nh lng VK trn mt vt bng c gi tr tng ng vi nh lng VK qua sinh thit [39]. Phntchm hc: quanst hnhnhvith m tnthngvnhnxts xm nhp ca vi sinh vt vo m [29]. 1.3.TNG QUAN V CHT LIU NGHIN CU 1.3.1.Ngun gc, cu trc ha hc, tnh cht EB12 l hn hp eleutherin v isoeleutherin vi t l 1:1, c chit xut v phn lp t thn hnh ca cy Sm i hnh Eleutherin subaphylla Granep. H La dn Iridaceae. Eleutherin v isoeleutherin cng c trong thn hnh ca mt s loi khc thuc chi Eleutherin [9], [24], [42]. V bn cht, Eleutherin v isoeleutherin l nhng naphtoquinon, c cu to ha hc nh sau: 20 Hnh 1.3: Cu to ha hc ca eleutherin v isoeleutherin. V tnh cht, chng c dng tinh th hnh kim, mu vng, nng chy 175-177C, tan tt trong ethyl acetat, aceton, cloroform v acid sulfuric c [5], [11]. 1.3.2.Tc dng Theo ng y, Sm i hnh c v ngt nht, tnh hi m, c tc dng t m dng huyt, ch huyt, ch khi [4], [9]. Dn gian dng Sm i hnh hm hay ngm ru lm thuc b, tr thiu mu, vng da, ho ra mu, thng tch lu huyt, Sm i hnh em phi kh, sao qua, hm ung lm thuc an thn, cha mt ng. Bt Sm i hnh dng ngoi cm mu, dng ung tr ho, lao hoc phi hp vi R qut ung tr ho, vim hng. Ngoi raSm i hnh gi nt, p ngoi da c tc dng cha mn nht, vim da, l nga, vy nn, t a, chc u. Mt s thnh phn ha hc ca Sm i hnh c phn lp v xc nh cu trc gm eleutherol, isoeleutherol, eleutherin, isoeleutherin [5], [9], [24], [38], [42], hongconin [62]. Trong , eleutherin v isoeleutherin c xc nh l hai thnh phn ch yu c hot tnh [42], [56]. Mt s nghin cu hot tnh sinh hc ca dch chit ethanol ca Sm i hnh, trong eleutherin v isoeleutherin l cc thnh phn chnh c hot tnh, cho thy c tc dng khng khun [33], [41], [42], [62], khng nm [51], chng virus [34], chng ung th, chng oxy ha , chng vim [56], cm mu [33] v c tc dng trn tim mch, tng cng lu thng mu mch vnh, c s dng lm thuc chng au tht ngc [34]. Tc dng khng khun c nhiu tc gi quan tm nghin cu. Cc nghin cu khc nhau u cho thy dch chit Sm i hnh c tc dng khng khun i vi Streptococcus spp., S. aureus, Bacillus spp., Shigella flexneri,..[41], 21 [42], [62]. S lng nghin cu v hot tnh sinh hc ca eleutherin v isoeleutherin c phn hn ch hn. Nm 1997, Hara H. v cng s m t tc dng c ch s nhn ln ca virus HIV trong H9 ca t bo lympho [38]. Nghin cu vo nm 2011 ca Tessele PB v cng s cho thy eleutherin v isoeleutherin c tc dng chng vim trn m hnh thc nghim gy vim bng carrageenin [56]. Kt qu nghin cu in vitro ca Su-Huyn Song (2009) cng cho thy isoeleutherin c tc dng chng vim do c ch tng hp nitric oxid trong m hnh gy vim bng liposacharid [54].22 Chng 2: I TNG V PHNG PHP NGHIN CU 2.1.NGUYN LIU, HA CHT, THIT B NGHIN CU 2.1.1.Mu nghin cuMu nghin cu EB12 do vin Ha hc v cc hp cht thin nhin, vin Khoa hc cng ngh Vit Nam cung cp, dng bt mn, mu vng. Mu nghin cu dng pha dung dch th hot tnh khng khun in vitro v bo ch kem EB12 th tc dng iu tr bng in vivo. Dungdchthhot tnhkhngkhunnng 50g/mlv 100g/ml. Cc dung dch sau khi pha c lc tit khun qua mng 0,02m. KemEB12:muthcbochthnhdngkemvicchmlng 100g/gv1000g/g.KemEB12cbochtibmnCngnghipdc, trng i hc Dc H Ni, theo phng php bo nh sau:Bo ch t dc kem EB12: thnh phn t dc kem EB12 gm 12,5% spermacetin, 12% sp trng, 56% parafin lng, 0,5% borat soda, 19% nc ct. Quy trnh bo ch t dc kem EB12: -Chun b pha du: un hn hp spermacetin, sp trng cho chy lng, sau trn ng nht vi parafin lng. -Chun b pha nc: ha tan borat soda vo nc ct. -Phi hp 2 pha: un nng pha du 60-65C, pha nc 65-70C, phi hp hai pha vi nhau v trn cho ng nht. Bo ch kem EB12: Nghin mn dc cht, phi hp vi t dc kem theo nguyn tc ng lng. 2.1.2.Ha cht, thuc th Thiopental natri: ca hng Rotex media, c. Kem sulfadiazin-bc 1% (bit dc Silivirin ca hng Satyam Pharm & Chemicals Pvt, n ). Mi trng thch thng: NaCl 0,5%; pepton 0,5%; cao tht 0,3%; thch 1,6%; nc va 100ml.23 V cc ha cht khc t tiu chun phng th nghim. 2.1.3.Thit b, my mc phc v nghin cu T nui cy vi sinh vt. Dng c th hot tnh khng khun. Dng c gy bng thc nghim. Dng c phn lp v nh lng vi khun. Dng c nghin cu m hc. 2.1.4.ng vt, vi sinh vt nghin cu 2.1.4.1. ng vt thc nghim Th, c 2 ging, cn nng 2,0-2,5 kg, nhanh nhn, mt sng, lng mt, khng mc bnh ngoi da. ng vt c nui n nh trong iu kin phng th nghim t nht 5 ngy trc khi nghin cu, cho n thc n dnh cho th v c cho ung nc sch trong sut qu trnh th nghim. 2.1.4.2. Cc chng VK, vi nm VK G(-)Escherichia coli ATCC 25922Proteus mirabilis BV 108 Shigella flexneri DT 112Samolnella typhi DT 220Pseudomonas aeruginosa VM 201 VK Gram (+) Staphylococcus aureus ATCC 1128 Bacillus pumilus ATCC 10241 Bacillus subtilis ATCC 6633 Bacillus cereus ATCC 9946Saracina lutea ATCC 9341 Vi nm:Candia albicanAspergilus sp. Mc xanh 24 Mc en 2.2.NI DUNG NGHIN CU Nghin cu tc dng c ch vi khun, vi nm in vitro ca EB12. NghincutcdngiutrtichcakemEB12trnbngnhitthc nghim. 2.3.PHNG PHP NGHIN CU 2.3.1.Nghin cu hot tnh khng khun, khng nm in vitro ca EB12 Phng php: nh gi hot tnh khng sinh bng phng php khuch tn trn thch. Nguyn tc: mu th c cho vo thch dinh dng cy vi sinh vt kim nh, hot cht t mu th khuch tn vo mi trng thch s c ch s pht trin ca vi sinh vt, to thnh vng v khun. Hot tnh khng khun ca mu th c nh gi da trn ng knh vng v khun. Mu th: dung dch EB12 50g/ml, dung dch Eb12 100 g/ml Dung dch khng sinh penicillin 28UI/ml Gentamycin 20g/ml Tin hnh: -Cy vi sinh vt kim nh vo mi trng thch dinh dng vi mt 106 t bo/100 ml mi trng. mi trng vo a petri, ch ng c. -cccgingthchckchthcbng nhautrnapetri,vtrccging cch xa nhau v cch xa thnh a petri. -Bsungccdungdchmuthvoccgingthchvithtch0,1ml/1 ging. - cc a petri 30C trong 18-24 gi. -c kt qu ng knh v khun ca cc mu th. nh gi kt qu: ng knh vng v khun c xc nh nh sau: D = Do d Trong : 25 D : ng knh vng v khun (mm). Do: ng knh vng ngoi ca vng v khun trn a thch (mm). d: ng knh ging thch (mm). 2.3.2. Nghin cu tc dng iu tr ti ch ca kem EB12 trn bng thc nghim 2.3.2.1. Trin khai m hnh gy bng nhit thc nghim Th nghim c thc hin ti phng th nghim b mn Dc lc, trng i hc Dc H Ni. Gy bng nhit cho th theo phng php c Pocidalo J.J (1955) v Hladovec J. (1961) m t vi dng c gy bng l bnh kim loi hnh tr rng, c th cha nc, c tay cm cch nhit. Th c co sch lng hai bn sng lng, gy m bng thiopental natri liu 25 mg/kg. Sau gy bng bng cch p bnh kim loi c nhit 100C vo da lng th di p lc 1kg, trong cc khong thi gian 35, 40 v 50 giy. Sau 3 ngy, quan st i th cc vt bng, ng thi ly mu m bnh hc ti m tn thng, quan st vi th, xc nh su tn thng, la chn thi gian gy bng thch hp tin hnh nghin cu. 2.3.2.2. NghincutcdngiutrtichcakemEB12trnbngthc nghim S nghin cu: Hnh 2.2: S nghin cu tc dng ca EB12 trn bng nhit thc nghim. -8 Gy bng -3071421 NgyNui n nh Xt nghim vi th o din tch vt bng Ly mu vi khun ti b mt vt bng Bi thuc Quan st, nh gi i th 26 Sau khi la chn thi gian gy bng thch hp, gy bng cho th nh m t mc 2.3.2.1, vi thi gian gy bng 35 giy,phn th thnh 4 l, nht ring mi con mt chung: L 1 (n=9): bi t dc kem EB12 (T dc). L 2 (n=10): bi kem sulfadiazin-bc 1% ( SSD). L 3 (n=10): bi kem EB12 100g/g. L 4 (n=9): bi kem EB12 1000/g. Tt c cc th u c bi mu th t sau khi gy bng 3 ngy n khi lin so, mi ngy 2 ln vo khong 8 gi sng v 16 gi chiu, lng mu th 0,03g/cm2/ln. Vt thng h, khng bng. Ch tiu theo di ti ch vt bng i th Tnh trng vt bng: sung huyt, ph n, tit dch, hoi t, s mc m ht, qu trnh biu m ha. Cc vt bng c theo di, ghi chp v nh gi hng ngy da trn thang im sau: Bng 2.1: Thang im nh gi tnh trng i th ti vt bng im0 im1 im2 im3 im Lng dch tit Kh t, khng c m t, c m trng rng vt bng KhngtTrung bnhNhiu Tnh trng lot Khng lot hoc trc vy, lin so Lot nng, kh hoc cha trc vy Lot nng, t Lot su, t Mc sung huyt KhngtTrung bnhNhiu Mc ph n KhngtTrung bnhNhiu Mc thu hp din tch vt bng: o din tch vt bng vo cc ngy trc khi bi mu th, ngy th 7, 14, 21 sau khi bi mu th bng giy bng knh v trng. 27 Mc thu hp din tch vt bng c tnh theo cng thc: Trong : X: mc thu hp din tch vt bng (%). S0: din tch vt bng lc cha bi mu th (cm2). St: din tch vt bng vo ngy th t sau khi bi mu th (cm2). Thi gian lin vt bng: c tnh t khi gy bng n khi lp vy bong hon ton, b mt vt bng c bao ph bi mt lp biu m mi. Vi th Hnh nh m bnh hc: Ly bnh phm ti m tn thng ti hai v tr: gia vt bng v ra vt bng vo ngy th 21 sau khi dng mu th. C nh mu bnh phm bng dung dch Bouin, vi trong paraffin. Ct lt dy 5-7mm, nhum tiu bn bng hematoxylin-eosin (HE). Quan st tiu bn di knh hin vi quang hc vi phng i 40, 100 v 400 ln, nhn xt s bin i t bo v cu trc m. Xt nghim vi khun v nm trn b mt vt bng Cc xt nghim VK v vi nm trn b mt vt bng c thc hin ti khoa Cn lm sng - Vin Bng Quc Gia, theo k thut ca Ivanov N.A v Danilova. Thi im ly mu: l ngy trc khi dng mu th (ngy th 3 sau khi gy bng, N0), ngy th 7 (N7), ngy th 14 (N14), ngy th 21 (N21) sau khi dng mu th. Phng php ly mu: Cc mu bnh phm ca cng mt vt bng nhng ngy khc nhau c ly ti cng mt v tr trn vt bng . S dng ming giy trong v khun c c sn l c din tch 1cm x 1cm, t ln b mt vt bng cha c lau ra. Dng tm bng v khun c lm m bng nc mui sinh l v khun, ln nh trn b mt vt bng, trong vng c l ca ming giy. Ln u, chm sao cho tm bng tip xc ht vi din tch tn thng bng trong vng 20s. Cho tm bng vo ng nghim c cha sn 2ml nc mui sinh l, lc nh trong vng 1 pht, y kn v mang i xt nghim. 28 nh loi vi khun: pha long dung dch trn vi cc t l khc nhau, ty theo s lng VK nhiu hay t. Dng micropipet ly 10l dung dch nc mui sinh l c cha VK pha long, cy ria u ln a mi trng thch dinh dng. mi trng 37C trong 24- 48 gi, cp kh bnh thng. Sau , da trn c im khun lc nh danh VK v vi nm. Xc nh s lng 2 loi VK ch yu vt bng: S. aureus, P. aeruginosa c trong 1 cm2 din tch vt bng: t mi VK mc ln mt khun lc, do d s VK ban u tng ng vi s khun lc. m s khun lc c trng ca tng loi VK, nhn vi t l pha long. 2.4.PHNG PHP X L S LIU S liu nghin cu c x l bng phn mm spss 16.0 Kt qu nghin cu c biu din di dng M SD (M: gi tr trung bnh, SD: sai s chun). So snh gi tr trung bnh gia cc l bng one-way ANOVA. Cc s liu khng thuc phn phi chun c kim nh bng test Kruskal Wallis, sau dng test Mann-Whitney U so snh gi tr trung bnh gia cc l. S khc bit gia cc l c ngha thng k khi p < 0,05. 29 Chng 3: KT QU NGHIN CU 3.1.TRIN KHAI M HNH GY BNG NHIT THC NGHIM Th c gy bng theo quy trnh m t, minh ha bng hnh nh di y: (a) (b)Hnh 3.1: Qu trnh gy bng thc nghim. (a)Loi b lng; (b) S dng ngun nhit; Theo di din bin ca cc vt bng tng ng vi cc khong thi gian gy bng 35, 40 v 50 giy cho thy: Tnh trng i th: sau khi gy bng, vt bng c mu trng ng, khong 1-2 gi sau bt u ph n, sung huyt (hnh 3.2a). Sau 3 ngy, cc vt bng bt u lot v xut hin hoi t t (hnh 3.2b). (a) (b)Hnh 3.2: Hnh nh vt thng sau khi gy bng (a): ngay sau khi gy bng; (b): 3 ngy sau khi gy bng Phn tch cu trc vi th m tn thng vo ngy th 3 sau khi gy bng cho thy cc vt bng u c nhng tn thng c trng nh hoi t vng trung tm, cc t bo vim tp trung ti vng tn thng, vim lan ta vng da xung quanh. Vi thi gian gy bng 35 giy, tn thng hoi t ti lp nng ca trung b, 30 mc tn thng khng trm trng. Vng ngoi tn thng khng ph n, khng c cc t bo vim xm nhp, da trong gii hn bnh thng (hnh 3.3). (a) (b)Hnh 3.3: Hnh nh vi th vt bng vi thi gian gy bng 35 giy (nhum HE) (a) Da vng lnh v tn thng (x40); (b) Da vng tn thng (x100) Vt bng vi thi gian gy bng 40 giy c hoi t da nng su ti cc nang lng ca lp trung b. Lp m lin kt vng tn thng xm nhp dy c bch cu a nhn. Vng ngoi tn thng c bch cu a nhn ri rc m lin kt trung b, phn ng vim ph n nh (hnh 3.4). (a) (b)Hnh 3.4: Hnh nh vi th vt bng vi thi gian gy bng 40 giy (nhum HE) (a): Da vng lnh v vng tn thng (x40); (b) Da vng tn thng (x400) Vi thi gian gy bng 50 giy, da b tn thng nng, hoi t su, bch cu a nhn xm nhp vo m m. Vng ngoi tn thng b sung huyt, ph n, xm nhp nhiu bch cu a nhn vo m lin kt trung b, phn ng vim ph va (hnh 3.5). 31 (a) (b)Hnh 3.5: Hnh nh vi th vt bng vi thi gian gy bng 50 giy (nhum HE) (a)Da vng lnh v vng tn thng (x40); (b) Da vng tn thng (x400) Nhn xt: mc tn thng tng theo thi gian gy bng. Gy bng thi gian 35 giy to ra vt bng III nng, vng da xung quanh khng c phn ng vim. Thi gian gy bng 40 giy to ra vt bng III su, da vng xung quanh tn thng vim nh. Vi thi gian gy bng 50 giy, vt bng to ra su IV, vim xm nhp ti lp m v c vn, vng da xung quanh c phn ng vim va. 3.2.TC DNG KHNG KHUN, KHNG NM IN VITRO CA EB12ng knh vng v khun ca cc mu th trong th nghim nh sau: Bng 3.1: ng knh vng v khun cc mu th i vi cc loi vi khun (mm) VK/ VNEB12 50g/mlEB12 100g/ml Penicillin28 UI/ml Gentamycin 20g/ml S. aureus9,26 2,5310,17 2,8120,470,42 Khng th B. subtilis6,01 1,295,65 0,16- Khng th B. pumilus7,10 1,627,61 1,00- Khng th B. cereus--16,220,12 Khng th S. lutea-- Khng th S. flexneri6,11 1,075,71 1,19Khng th12,340,61 P. mirabilis5,71 1,425,77 1,24Khng th17,270,42 E. coli--Khng th15,400,47 S. typhi--Khng th17,500,33 P. aeruginosa--Khng th10,651,27 C. albican--Khng th Khng th Aspergic sp.--Khng th Khng th Mc xanh--Khng th Khng th Mc en--Khng th Khng th Nhn xt: dung dch mu th EB12 cc nng 50g/ml v 100g/ml c tc 32 dng c ch s pht trin ca cc vi khun Bacillus subtilis, S. aureus, Bacillus pumilus, Bacillus pumilus, Shigella flexneri, Proteus mirabilis,tuy nhin ng knh vng v khun nh hn ng knh vng v khun ca khng sinh chng tng ng, khng c tc dng c ch i vi cc vi khun v vi nm: E.coli, Samolnella typhi, P. aeruginosa, Bacillus cereus, Saracina lutea, C. albican, Aspergic sp. , nm Mc xanh, nm Mc en. 3.3.TCDNGIUTRTICHCAKEMEB12TRNBNG NHIT THC NGHIM 3.3.1.Tnh trng chung ca th Ngy u sau khi gy bng, th mt, t hot ng, thng nm yn, n ung km. Vi ngy sau, th nhanh nhn dn, n ung tt hn. Khong 2 tun sau khi iu tr, th nhanh nhn, n ung v hot ng bnh thng. 3.3.2.Din bin i th ti vt bng 3.3.2.1. Din bin chung Ngay sau khi gy bng, vt bng c mu trng ng, khng phng rp, c ranh gii r rng vi vng da lnh. Khong 1-2 gi sau, ra xung quanh vt bng nhn r qung sung huyt. Ngy th 3 sau khi gy bng, vt bng bt u lot v xut hin hoi t t. Sau khi bi mu th 7 ngy, cc vt bng vn t, c m trng, cha thy c s khc bit gia cc l. Mt s hnh nh vt bng vo ngy th 3 sau khi gy bng v ngy th 7 iu tr c trnh by hnh 3.6. Sau 14 ngy, a s vt bng co li, gim tit dch, mt s vt bng cn t v c m. Mt s vt bng l EB12 1000g/g bt u rng m hoi t. Sau 21 ngy iu tr, cc vt bng ht m, kh, din tch c thu hp ng k. Mt s vt bng thuc l sulfadiazin-bc 1% v l EB12 1000g/g bt u bong vy v lin so. Mt s hnh nh vt bng sau khi bi mu th 14 v 21 ngy c minh ha hnh 3.7. 33 Ngy 0Ngy 7 L t dc L SSD L EB12 100g/g L EB12 1000g/g Hnh 3.6: Hnh nh cc vt bng vo ngy th 0 v th 7 iu tr 34 Ngy 14Ngy 21 L t dc kem EB12 L SSD L EB12 100g/g L EB12 1000g/g Hnh 3.7: Hnh nh cc vt bng vo ngy th 7 v th 14 iu tr 35 3.3.2.2. nh gi chi tit tnh trng i th vt bng Cc vt bng c theo di, nh gi hng ngy. Kt qu thu c c th hin bng sau: Bng 3.2: Kt qu nh gi din bin i th ti vt bng (im) LMu thN0 N7 N14 N21 1T dc (n=9)9,12 1,7210,50 0,751,75 0,891,13 0,99 8,60 1,9510,00 0,811,80 0,791,00 0,82 2 Sulfadiazin-bc 1% (n=10)p = 0,63p = 0,24p = 0,97p = 0,90 9,00 1,4910,00 0,811,80 0,921,20 0,42 3 EB12 100g/g (n=10)p = 0,89p = 0,24p = 0,76p = 0,70 9,00 2,0710,00 0,751,50 0,530,87 0,83 4 EB12 1000g/g (n=9)p = 0,88p = 0,23p = 0,44p = 0,72 Trong p: s khc bit so vi l bi t dc kem EB12. Nhn xt:-Ti thi im cha iu tr (N0): tnh trng cc vt bng cc l ging nhau (p> 0,05). -Trong 7 ngy u iu tr, cc vt bng u din bin nng dn ln, lot su v rng,tngdchtit,tngm.imnhgimcbngcclutngtuy nhin khng c s khc bit gia cc l. -Ngy th 14 sau khi iu tr (N7), tnh trng cc vt bng c ci thin ng k, tuy nhin cha c s khc bit gia cc l. -nh gi tnhtrng i th vt bngvongyth21sau khi iutr(N21)cho thy cc vt bng l EB12 1000g/g c ci thin hn so vi l bi t dc (0,87 so vi 1,13), tuy nhin s khc bit cha t mc ngha thng k (p> 0,05). 3.3.2.3. Mc thu hp din tch vt bng Din tch vt bng c o vo cc ngy: trc khi iu tr (N0), ngy th 7 (N7), ngy th 14 (N14) v ngy th 21 (N21) sau khi iu tr. Mc thu hp din tch vt bng (%) cc ngy N7, N14 , N21 so vi trc khi iu tr c th hin 36 bng sau: Bng 3.3: Mc thu hp din tch vt bng LMu thnN7 N14 N21 1T dc 9X(%)34,29 13,3062,96 13,4089,02 8,04 X(%)30,99 9,6367,04 17,2086,49 12,05 2SSD10 pp = 0,67p = 0,54p = 0,59 X(%)36,22 19,1067,33 11,3985,99 10,89 3 EB12 100g/g 10 p0,80p = 0,51p = 0,52 X(%)27,88 19,971,12 12,990,42 9,48 4 EB12 1000g/g 9 pp = 0,42p = 0,24p = 0,81 Trong p: s khc bit khi so vi l bi t dc. Kt qu bng trn cho thy din tch vt bng c thu hp dn trong qu trnh iu tr. Sau 21 ngy dng thuc, din tch cc vt bng c thu hp 85-90%. Tuy nhin mc thu hp din tch vt bng cc l khng c s khc bit (p> 0,05). 3.3.2.4. Thi gian lin vt bng Cc vt bng c quan st v ghi chp din bin hng ngy. Vt bng c coi l lin hon ton khi bong lp vy pha trn, b mt vt bng c bao ph bi mt lp biu m mi. Thi gian lin vt bng trung bnh ca cc l c th hin bng sau: Bng 3.4: Thi gian lin vt bng LMu thNThi gian lin vt bng (ngy) P 1T dc kem EB12925,00 1,06 2Sulfadiazin-bc 1%1023,37 2,700,60 3EB12 100g/g1025,42 1,390,98 4EB12 1000g/g924,33 3,380,99 Trong p: s khc bit so vi l bi t dc kem EB12. Kt qu trn cho thy thi gian lin vt bng l bi sulfadiazin-bc 1% v l bi EB12 1000g/g c gim so vi l bi t dc kem EB12, tuy nhin s khc bit 37 khng c ngha thng k (p>0,05). 3.3.3.Din bin cu trc vi th ti vt bng Mt s hnh nh vi th ti vt bng vo ngy th 21 sau khi iu tr: (a) (b) (c)(d) Hnh 3.8: Hnh nh m ht ti to, c vim mn tnh ( nhum HE) (a) L t dc kem EB12 (x400)(b)L SSD (x400) (c) L EB12 100g/g (x100)(d) L EB12 1000g/g (x400) (a) (b) Hnh 3.9: Hnh nh tn thng bng ang ti to (nhum HE, x400) (a)Lp thng b nng, c t bo vim thm nhp l SSD (b)Nang lng v tuyn m hi di lp thng b vt bng bi EB12 1000g/g Hnh nh vi th ti vt bng cho thy: cc m tn thng u cn tn ti cc t bo vim nh: bch cu a nhn, bch cu n nhn, i thc bo, m bo. M ht 38 in hnh vi mch mu tn to, si lin kt v cc t bo x non xut hin cc vt bng. Cc t bo lp thng b c hnh thnh cc vt bng thuc l sulfadiazin bc 1%, EB12 100g/g, EB12 1000g/g, tuy nhin cha xut hin l bi t dc kem EB12. 3.3.4.Din bin ca qun th vi khun ti vt bng 3.3.4.1. Cc loi vi khun phn lp c ti vt bng T l cc VK phn lp c qua cc ln cy khun: Bng 3.5 : T l cc vi khun phn lp c ti vt bng VKS ln phn lp c% t l phn lp c (%) S. aureus7573,53 P. aeruginosa1716,67 E. coli32,94 E. cloacea43,92 K. pneumoniae21,96 Ent. Faecium21,96 Str. Agalactiae21,96 Bacillus spp.32,94 Pro. Mirabillis21,96 Aci. Lowfii32,94 Nhn xt: kt qu trn cho thy cc loi VK phn lp c nhiu nht l S. aureus 73,53%, P. aeruginosa 16,67% v cc trc khun ng rut (E. coli, E. cloacea, K. pneumoniae ) 8,82%. 3.3.4.2. Din bin s lng loi vi khun gy bnh ch yu ti vt bng Cc loi VK gy bnh ch yu vt bng l S. aureus, P. aeruginosa. Qua cc ln cy khun cc thi im: 3 ngy sau khi gy bng (N0), 7 ngy sau khi iu tr (N7) v 14 ngy sau khi iu tr (N14), t l xut hin ca cc VK trn l S. aureus (73,53%), P. aeruginosa (16,67%). S liu v P.aeruginosa khng x l thng k. S lng VK S. aureus qua cc ln ly mu nh sau: Bng 3.6: S lng vi khun S. aureus trn 1 cm2 din tch vt bng ( Vk/cm2) 39 LMu thNN0 N7 N14 1T dc 91412,00 831,701185,00 358,80166,00 61,30 1437,00 839,50161,30 62,80113,20 31,30 2SSD10 p2-1 = 0,713p2-1 = 0,001p2-1 = 0,635 842,00 545,94477,00 396,70167,00 41,00 p3-1 = 0,368p3-1 = 0,007p3-1 = 0,7923 EB12 100g/g 10 p3-2 = 0,631p3-2 = 0,739p3-2 = 0,315 2112,80 1055,60254,38 127,01202,70 89,25 p4-1 = 0,662p4-1 = 0,020p4-1 = 0,950 p4-2 = 0,360p4-2 = 0,965p4-2 = 0,633 4 EB12 1000g/g 9 p4-3 = 0,146p4-3 = 0,829p4-3 = 0,897 Trong : px-y: s khc bit v s lng VK l x so vi l y. Kt qu trn cho thy: -S lng VK S. aureus trn 1cm2 din tch vt bng ti thi im trc khi iu tr cc l tng ng nhau (p> 0,05). -Saukhiiutr,slngVKcclsulfadiazin-bc1%,EB12100g/gv EB12 1000g/g gim nhanh hn l bi t dc. S khc bit t ngha thng k tithiim7ngysaukhibimuth(p0,05),dotc dngkhngkhuncaEB12100g/gvEB121000g/gtngngvi sulfadiazin-bc 1%. Vy kem EB12 hm lng 100g/g v 1000g/g c tc dng khng khun trn S. aureus khi iu tr ti ch vt bng, tc dng khng khun ny tng ng kem sulfadiazin-bc 1%. 40 Chng 4: BN LUN 4.1.V M HNH NGHIN CU 4.1.1.La chn m hnh nghin cu Trong s cc tc nhn gy bng thc nghim, nhit c u im hn so vi dng in v ha cht bi d dng kim sot su v din tch vt bng, c th to ra nhng tn thng ng nht [53]. Hn na, cc m hnh bng nhit p dng c trn nhiu loi ng vt, dng c n gin, d tin hnh [53]. Trn thc t, bng nhit hay gp nht, chim 84-94% cc nguyn nhn gy bng [17], [18], cha k bng ha cht v bng in cng c mt phn c ch do nhit. y l nhng l do chng ti quyt nh tin hnh nghin cu trn m hnh bng nhit thc nghim. Trong s cc phng php gy bng nhit thng dng, phng php s dng bnh kim loi to ra c nhng tn thng ng nht, c th ty chn cc thng s trong qu trnh gy bng to ra vt bng c din tch v su mong mun [53]. Hn na phng php ny c dng c n gin, d tin hnh, c th trin khai trn nhiu loi ng vt khc nhau [53]. nc ta, phng php ny c p dng thnh cng trong cc nghin cu ca Nguyn Th T (1989) [22], V Th Ngc Thanh (2003) [14] v Trn Th L (2011) [10]. 4.1.2.La chn thi gian gy bng Trn c s cc cng b trc y ca Nguyn Th T (1989) [22], V Th Ngc Thanh (2003) [14] v Trn Th L (2011) [10] tin hnh gy bng ti Hc vin Qun Y, chng ti thit k dng c gy bng c cu to tng t dng c gy bng ca Hc Vin Qun Y, s dng cho m hnh gy bng nhit 100C di p lc 1 kg. ng thi, chng ti tin hnh th nghim la chn thi gian tip xc thch hp to ra tn thng bng c din tch v su nh mong mun. Nhit gy bng c c nh 100C. Dng c gy bng c lm nng bng cch ngm vo b cch thy. Nh c nhit dung ring ln, nc bn trong bnh kim loi gip cho nhit ca dng c hng nh trong sut thi gian gy bng. 41 p lc ln vng da gy bng c c nh mc 1kg, s dng chnh trng lng ca dng c gy bng. Trong qu trnh thc hin, k thut vin ch tc dng lc gi dng c gy bng tip xc vi da, do p lc ln da ng khi gy bng cc vt bng ng u, khng ph thuc mun ch quan ca k thut vin. Thi gian: ng vt c gy bng trong nhng khong thi gian 35 giy, 40 giy v 50 giy.Kt qu phn tch tnh trng i th v hnh nh vi th ti vt thng bng vo ngy th 3 sau khi gy bng cho thy: Gy bng thi gian 35 giy to ra vt bng III nng, vng da xung quanh khng c phn ng vim. Thi gian gy bng 40 giy to ra vt bng III su, da vng xung quanh tn thng vim nh. Vi thi gian gy bng 50 giy, vt bng to ra su IV, hoi t su, vim xm nhp ti lp m v c vn, vng da xung quanh c phn ng vim va. Vi mc ch to ra nhng vt bng su III phc v nghin cu, chng ti la chn thi gian gy bng l 35 giy to ra tn thng c su ph hp, ng thi khng lm nh hng n tnh trng ton thn ca ng vt th nghim. 4.2.V TC DNG KHNG KHUN, KHNG NM IN VITRO CA EB12Kt qu nghin cu in vitro cho thy dung dch EB12 nng 50 g/ml v 100 g/ml c tc dng c ch s pht trin ca cc VK Bacillus subtilis, Staphylococcus aureus, Bacillus pumilus, Shigella flexneri v Proteus mirabilis, khng c tc dng c ch i vi cc vi khun v vi nm: E.coli, Samolnella typhi, P. aeruginosa, Bacillus cereus, Saracina lutea, C. albican, Aspergic sp. , nm Mc xanh, nm Mc en. Kt qu ny ph hp vi kt qu ca mt s tc gi trong v ngoi nc: Ifesan v cng s (2009) nghin cu hot tnh khng khun ca dch chit ton phn v cc phn on dch chit ca E. americana trong eleutherin v isoeleutherin l cc thnh phn c hot tnh chnh. Kt qu cho thy cc dch chit ny c tc dng c ch s pht trin ca cc VK B. subtilis, S. aureus v mt s VK khc nh B. cereus, B. licheniformis , khng c tc dng trn P. aeruginosa v E. coli [41], [42]. Nghin cu ca Hunh Kim Diu cho thy dch chit ton phn ca Sm i 42 hnh tc dng khng khun i vi cc VK S. aureus, S. faecalis, Aeromonas hydrophila, tc dng yu hn trn P. aeruginosa v yu nht trn E. coli [5]. Kt qu ny ph hp vi kt qu ca chng ti, mu th EB12 cc nng 50g/ml v 100g/ml khng th hin tc dng c ch i vi P. aeruginosa v E. coli trong khi c ch r rt S. aureus. Mt s nghin cu khc cng cho thy dch chit ca Sm i hnh c tc dng khng khun i vi cc VK tng t [9], [11], [24], [62]. Tc dng khng khun ca eleutherin v isoeleutherin c khng nh qua nhiu nghin cu. C ch khng khun c th gii thch da trn cu trc phn t ca eleutherin v isoeleutherin, vn c bn cht l nhng naphtoquinon [25], [42]. Naphtoquinon l nhm cc cht chuyn ha th cp c mt rng ri trong t nhin, nhiu h thc vt, nhiu loi vi sinh vt nh VK v nmvi nhiu vai tr sinh l khc nhau [25]. Chng l nhm cc cht c nhiu hot tnh dc l nh chng vim, khng khun, khng nm, gy c t bo, do c kh nng hot ng nh nhng cht c ch vn chuyn in t, ngn cn phosphoryl oxy ha, bt hot cc acid amin[25], [40]. Nhng thc vt c cha naphtoquinon thng c s dng rng ri trong y hc dn gian v gn y ngy cng c nghin cu s dng nhiu trong y hc hin i. C ch khng khun ca eleutherin v isoeleutherin c mt s tc gi gii thch da trn c ch tc dng chung ca naphtoquinon nh sau: -Cnhtranhvnchuynintviccthnhphntrongmngtbo,dnn thay i vn chuyn cc ion qua mng, thay i cc c ch vn chuyn tch cc qua mng [40]. -To phc khng phc hi vi cc nhm amin trong protein, do lm thay i cu trc v chc nng ca cc protein trong t bo [46], [40]. -KthpviADN,cchtnghpARNm,dongncntnghpprotein [25], [40]. 43 4.3.V TCDNGIU TR TI CHCAEB12 TRN BNGNHIT THC NGHIM 4.3.1.Tc dng khng khun khi iu tr ti ch vt bng Kt qu nghin cu in vitro cho thy EB12 c tc dng c ch s pht trin ca mt s VK. Trn c s , chng ti quyt nh nh gi tc dng khng khun ca EB12 khi iu tr ti ch vt bng. nh gi c tc dng khng khun ca thuc khi iu tr ti ch bng nhit thc nghim, ng vt c s dng trong nghin cu l th. Th l ng vt rt d b nhim khun, c th dng cho cc nghin cu v nhim khun bng m khng cn cy thm VK vo vt bng [53]. Tuy nhin, chnh v c tnh d b nhim khun, th cng yu cu cc iu kin v sinh nghim ngt trong qu trnh th nghim. Trong th nghim ca chng ti, trc khi gy bng, th c co lng hai bn sng lng mt cch cn thn, khng lm xc da trc khi gy bng. Cc vt bng c to ra trn vai th, t tip xc vi chung nui, do m bo v sinh hn. Trong qu trnh iu tr cho th, cc k thut vin s dng gng tay v khun, mi gng tay ch bi mu th cho mt vt bng, mc ch ngn nga ly nhim cho VK gia cc vt thng. Cc iu kin v sinh c tun th nghim ngt trong sut qu trnh nghin cu nhm nh gi ng tc dng khng khun ca mu th. 4.3.3.1. Qun th vi khun ti vt bng Trong nghin cu ca chng ti, qua cc ln cy khun cc thi im: trc khi dng thuc, ngy th 7 v ngy th 14 dng thuc, cc loi VK xut hin nhiu nht l: S. aureus (73,53%), P. aeruginosa (16,67%) v cc loi trc khun ng rut (E. coli, E. cloaceae, K. pneumoniaceae) (8,82%). Kt qu ny ph hp vi mt s nghin cu khc v cn nguyn gy nhim khun bng. Theo nghin cu ca nhiu tc gi, cc loi VK ch yu gy nhim khun bng l S. aureus, P. aeruginosa v cc trc khun ng rut [7], [20], [29], [31]. Nguyn Nh Lm v L c Mn (2010) phn lp VK t 725 mu bnh phm thy cc VK gy bnh ch yu l P. aeruginosa 39,37%, S. aureus 32,15% v Aci. 44 baumanii 17,14%. Trong nhiu vt bng c s kt hp ca nhiu loi VK [7]. C. Glen Mayhall (2003) thng k kt qu phn lp VK t 2501 vt bng t 1974-1998 cho thy S. aureus chim 23,0%, P. aeruginosa chim 19.9%, tip n l E. coli v cc loi Pseudomonas [47]. Theo L Th Kim Anh v Thu Hng (1997), cc VK hay gp nht m ti vt bng l S. aureus (27,4%), P. aeruginosa (25,8%) tip theo n Alcailgenes v cc loi Pseudomonas chim 13,5% [2]. Tuy nhin, trong cc mu bnh phm l mu, P. aeruginosa hay gp hn, t l phn lp c l 26,22% trong khi S. aureus thp hn, chim khong 13,11% [2].Cc VK thng gp vt bng u l nhng loi VK gy bnh nguy him, c c lc cao, c kh nng xm nhp su vo c th, gy nhim khun huyt [7], [42]. Hu ht cc loi VK phn lp c ti cc vt bng u khng li hu ht cc khng sinh thng dng [2], [7]. Kh nng khng thuc ca chng ngy cng lan rng thng qua cc plasmid cha gen khng thuc [47]. V vy, vic nghin cu tm ra thuc c tc dng tt trn cc VK thng gp, t b khng, c ngha rt ln i vi cng tc iu tr bng. 4.3.3.2. Tc dng khng khun khi iu tr ti ch ca EB12 Mc v nguy c nhim khun c th nh gi da trn chng loi v mt VK trn b mt vt bng hoc trong m ca tn thng bng [47]. Vt bng cng c cc loi VK c kh nng xm nhp su hoc VK vi s lng ln th cng c nguy c nhim khun huyt, nhim khun ton thn. V th, nu thuc c th lm gim s lng VK ti vt bng s ngn nga c nhng bin chng nng n ca nhim khun bng, to iu kin qu trnh lnh vt thng xy ra. Trong nghin cu ca chng ti, S. aureus l VK xut hin nhiu nht. thi im cha iu tr, c ti 23/38 vt bng c s c mt ca S. aureus, tng ng 60,5%. Trong c ba ln cy khun vo ngy th 0, th 7 v th 14 iu tr, tn xut xut hin ca S. aureus ln ti 73,53%. Trong thc t iu tr bng, S. aureus cng l VK thng gp nht, l nguyn nhn chnh gy nhim khun vt bng, l nguyn nhn hay gp gy nhim khun huyt trong bng [7], [20], [29], [31], [47]. S.aureus 45 l loi VK nguy him, c c ni c t v ngoi c t, khi xm nhp vo c th gy ra nhiu bin chng nng n [42]. iu tr lm gim s lng S. aureus ti vt bng s gip ngn nga nhim khun vt bng, nhim khun huyt, to iu kin cho qu trnh lin vt thng din ra. V vy, chng ti nh gi tc dng khng khun ca kem EB12 trn vt bng thng qua din bin ca S. aureus trong qu trnh iu tr. Ti thi im trc khi iu tr, s lng VK S. aureus trn 1cm2 din tch vt bng cc l SSD, l EB12 100 g/g v l EB12 1000 g/g tng ng vi l bi t dc (p > 0,05).Sau khi bi mu th, s lng VK cc l u c xu hng gim xung, tuy nhin c s khc bit gia cc l. ngy th 7 sau khi iu tr, s lng VK cc l SSD, l EB12 100g/g v l 1000 g/g gim nhanh r rt so vi l bi t dc (p 0,05). Do c th kt lun rng tc dng khng khun trn S. aureus ca EB12 cc nng 100g/g v 1000g/g tng ng vi sulfadiazin bc 1%. Nhng kt qu trn cho thy EB12 c tc dng khng khun i vi S. aureus khi iu tr ti ch. Tc dng khng khun ca EB12 cc nng 100 g/g v 1000g/g tng ng vi sulfadiazin bc 1%. Cha c nhiu nghin cu nh gi tc dng khng khun eleutherin, isoeleutherin hoc dch chit Sm i hnh khi iu tr ti ch. Tuy nhin c nhiu nghin cu in vitro cho thy kh nng c ch S. aureus ca eleutherin v isoeleutherin. Mt khc, trong dn gian, Sm i hnh c s dng ph bin nh mt thuc p ngoi iu tr mn nht, l lot,iu ny ph hp vi nghin cu ca chng ti v kh nng khng khun ca thuc EB12 khi iu tr ti ch. Nghin cu ca Ifesan v ng tc gi (2009) cn ch ra rng dch chit ca E. 46 american vi eleutherin v isoeleutherin l nhng thnh phn c hot tnh chnh, khng ch c tc dng c ch s pht trin ca S. aureus m cn c tc dng bt hot cc enzym ngoi bo v c ch sn xut cc ni c t ca S. aureus ngay c nng MIC, thm ch MIC [42]. iu ny c ngha quan trng i vi thuc iu tr ti ch vt thng bng bi s c mt ca S. aureus thng gia tng kh nng xm nhp ca cc VK khc do tc dng phn gii m ca cc enzym ngoi bo l protease v lipase [42]. 4.3.2.Tc dng chng vim, lm sch vt bng Ngay sau khi b tn thng, ti v tr vt thng c phn ng vim cp. y l phn ng t nhin ca c th nhm ngn nga s lan rng ca cc tc nhn gy hi n cc m ln cn, loi b cc mm bnh cng nh cc t bo cht ca c th, to iu kin cho qu trnh lin vt thng xy ra [1]. Tuy nhin, nu p ng vim ca c th xy ra qu mc s gy tn thng m th pht, lm chm ch qu trnh lin vt thng, thm ch gy ra nhng ri lon ton thn. V vy nu thuc iu tr ti ch c tc dng ln qu trnh vim, ngn cn phn ng vim qu mc s to iu kin thun li cho qu trnh lin vt thng xy ra [57]. nh gi mc vim ca vt bng, chng ti da trn nhng c trng c bn ca phn ng vim cp nh: sung huyt, ph n, tit dch, lot. Cc ch tiu c nh gi bng phng php cho im t 0 n 3 im th hin cho mc nng ca phn ng vim. Tnh trng vim ca vt bng c nh gi da trn tng im ca 4 ch tiu trn, phn ng vim cng mnh, tng im cng cao. Kt qu theo di, nh gi v cho im hng ngy ca cc vt bng cho thy: Ti thi im bt u iu tr, cc vt bng ph n, sung huyt, bt u lot v c biu hin c hoi t t. Cc im s cho thy khng c s khc bit v mc bng gia cc l (p>0,05). iu ny chng t qu trnh gy bng v phn l l tng i ng u. Ti thi im 7 ngy sau khi iu tr, cc vt bng vn chy dch. vng khng lot, cc cht tit v m hoi t to thnh lp vy dy, bn di lp vy c nhiu m trng. nh gi cho thy mc vim cc l u tng ln. y l din bin 47 sinh l bnh thng ca giai on vim cp sau bng. ng thi cng ph hp vi kt qu phn lp VK ti vt bng m chng ti trnh by phn trc. S c mt ca VK ti vt bng thc y phn ng vim xy ra mnh m hn. Nhng quan st sau cho thy mc vim gim dn tt c cc l, cc vt bng khng cn sung huyt v ph n, gim dn dch tit, cc vt bng co li dn. Ti thi im 21 ngy sau khi iu tr, cc vt bng l chng dng c mc bng thp hn so vi l bi t dc, tuy nhin s khc bit cha t mc ngha thng k. L EB12 1000g/g cho thy s gim mc bng so vi l bi t dc cc thi im 14 v 21 ngy sau khi iu tr, tuy nhin s khc bit cng cha t mc ngha thng k. Kt qu trn cho thy, kem EB12 cc hm lng 100g/g v 1000g/g dng ti ch cha th hin tc dng chng vim, lm sch vt bng mt cch r rt. Thc t, cha c nghin cu nh gi tc dng chng vim ti ch ca eleutherin, isoeleutherin hoc dch chit Sm i hnh. Tuy nhin mt s nghin cu cho thy eleutherin isoeleutherin c tc dng chng vim cp khi dng ng tim tnh mch hoc ng ung. Nghin cu ca Tessele PB v cng s nm 2011 cho thy tim tnh mch eleutherin hoc isoeleutherin vi liu t 1,04 n 34,92 mmol/kg c tc dng gim ph chn chut trong m hnh gy ph thc nghim bng carrageenin, tc dng ca chng tng ng vi indomethacin tim ng tnh mch vi liu 13,90 mmol/kg [56]. Cng trong nghin cu ny, isoeleutherin liu 34,92 mmol/kg dng ng ung c tc dng gim ph chn chut trong m hnh gy vim bng carrageenin, tc dng ny tng ng indomethacin liu 13,90 mmol/kg dng ng ung. Tuy nhin, eleutherin dng ng ung khng c tc dng gim ph chn chut. iu ny gi rng tc dng ca eleutherin v isoeleutherin khc nhau v tc dng ca chng ph thuc vo ng dng. S khc nhau ny c th lin quan n cu hnh quang hc v s hp thu cc cht.Nghin cu ca Su-Hyun Song (2009) cng cho thy isoeleutherin c tc dng 48 chng vim in vitro do c ch tng hp nitric oxide trong m hnh gy vim bng lipopolysaccharide [54]. S khc bit v kt qu nghin cu ca chng ti vi cc nghin cu khc c th do liu lng v ng dng khc nhau. 4.3.3.Tc dng trn qu trnh lin vt bng Thi gian lin vt bng Mt trong nhng mc tiu iu tr bng l y nhanh qu trnh qu trnh lin vt thng, rt ngn thi gian lin vt bng. Trong nghin cu ca chng ti, cc vt bng c theo di v quan st hng ngy. Vt bng c coi l lin hon ton khi lp vy pha trn bong trc ra hon ton, b mt bng c bao ph bi mt lp biu m mi. Thi gian lin vt bng trung bnh ca cc l t dc, SSD, EB12 100g/g, EB12 1000g/g ln lt l 25,00; 23,37; 25,42; 24,33 ngy. Thi gian lin vt bng l bi SSD v EB12 1000g/g c gim so vi l bi t dc, tuy nhin s khc bit khng c ngha thng k (p> 0,05). Mc thu hp din tch vt bng Khi nh gi qu trnh lin vt bng, mc thu hp din tch vt bng l mt trong cc ch tiu c quan tm. Din tch vt bng thu hp li cho thy qu trnh vim khng cn lan rng v gy tn thng m xung quanh, nguy c nhim khun cng gim xung. Ti thi im trc khi iu tr, din tch trung bnh ca cc vt bng cc l tng ng nhau (p>0,05), chng t qu trnh gy bng v phn l kh ng u. Theo di din bin trong sut qu trnh lin vt thng cho thy khng c s khc bit gia cc l c v din tch vt bng ln t l thu hp din tch vt bng (p>0,05). Din bin cu trc vi th ti vt bng Hnh nh vi th m tn thng vo ngy th 21 iu tr cho thy cc vt bng u c s hnh thnh m ht in hnh vi nhiu mch mu tn to, nhiu si lin kt cng nh cc t bo x non. Vng tn thng ca cc vt bng u tp trung 49 nhiu t bo vim, vi phn ng vim mn tnh. Kt qu trn vi th cho thy khng c s khc bit gia cc vt bng cc l. Kt qu nghin cu ca chng ti cho thy eleutherin v isoeleutherin cha th hin tc dng y nhanh qu trnh lin vt thng bng. Qu trnh lin vt thng da l mt qu trnh phc tp, gm nhiu giai on, chu nh hng bi nhiu yu t [1]. Cc thuc tc dng ln qu trnh lin vt thng da thng tc ng thng qua mt hoc nhiu c ch sau: khng khun, chng vim, chng oxy ha, lm sch vt thng, to pH ti ch thun li cho qu trnh lin vt thng xy ra; tng cng tng hp protein, collagen; tng cng di c collagen, t bo sng, n nh mng mi to ra [57]. Theo kt qu ca mt s nghin cu c cng b, eleutherin v isoeleutherin c tc dng chng vim [56], [54], chng oxy ha [38], khng khun [6], [33], [41], [42], [62], iu ny gi rng c th eleutherin v isoeleutherin c th c tc dng ln qu trnh lin vt bng. Tuy nhin, nh gi nh hng ca mt cht ti ton b qu trnh lin vt thng cn tin hnh cc nghin cu in vivo [57]. Trong nn y hc dn gian ca nhiu nc, Sm i hnh v dch chit ca n c dng kh ph bin iu tr cc vt thng ngoi da [4], [9]. Tuy nhin c rt t cng trnh nghin cu v tc dng ca dch chit Sm i hnh hoc cc hot cht ca n trn qu trnh lin vt thng da. Nm 1997, trong mt nghin cu nh gi tc dng trn qu trnh lin vt thng ca mt s tho dc truyn thng Peru, Leon F.V v cng s kt lun rng dch chit E. bulbosa khng c tc dng ng k trn qu trnh lin vt thng [59]. Aadesh v cng s (2013) nghin cu tc dng lm lin vt thng ca dch chit methanol ca E. indica thy c tc dng kch thch qu trnh lin vt thng thng qua tng tng hp hydroxyproline, tuy nhin thnh phn ca dch chit khng bao gm eleutherin v isoeleutherin m ch c cc steroid, flavonoid, terpen, tannin v cc alcaloid [58]. 50 KT LUN T kt qu nghin cu v tc dng iu tr ca EB12 trn bng thc nghim, chng ti xin php c rt ra mt s kt lun sau: 1. EB12ctcdngkhngkhuninvitro:dungdchEB1250g/ml,100g/mlctc dngcchsphttrincaccvikhun:Bacillussubtilis,Staphylococcusaureus, Bacillus pumilus, Shigella flexneri v Proteus mirabilis. 2. TcdngiutrtichcakemEB12100g/gv1000g/gtrnbngnhitthc nghim: Kem EB12 100 g/g v 1000 g/g c tc dng khng khun i vi S. aureus khi iu tr ti ch. Tc dng khng khun ca kem EB12 100g/g v 1000g/g khi dng ti ch tng ng vi kem sulfadiazin bc 1%. KemEB12 100g/gv 1000g/gchath hintc dngchngvim,lmschvt bng khi iu tr ti ch vt thng bng nhit thc nghim trn th. 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