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ElEstudioExperimentalsobreelEfectodelFRC001(LíquidoTinaPianChinaNo.1)sobrelaInmunidadInespecíficade

Ratones

RobertW.BradfordD.Sc.1KexiangDing2

1.BradfordResearchInstitute,CA,USAProfessorofCapitalUniversity,WashingtonDC

2.FRCFreeRadicalBiology&MedicalResearchCenter

ABSTRACTOEstetrabajoinvestigóelefectodelFRC001sobrelainmunidadinespecíficaderatonesylareaccióninjertoversushuésped(GVHR).LosresultadosdemuestranqueelFRC001tuvociertoefectosobrelafunciónfagocíticadelmacrófagomononuclear,elíndicedelbazoyelíndicedeltimo,transformaciónlinfocitariadelbazo,formaciónhemolizantedelsuero,elcoeficientedelareaccióndelbazodelhuéspedycoeficientedeestimulaciónyelíndicedeestimulación,porejemploFRC001puedeaumentaryayudarlainmunidadhumoralycelularenunabaseexperimentalparaelusodelFRC001pararegularlainmunidadyprevenirycurartumores.PalabraClave:FRC001InmunidadInespecíficadelareaccióninjertoversushuésped(GVHR)

HISTORIALaformacióndetumoresserelacionaaladeficienciainmunológica.Enlosúltimosañoshahabidomuchosreportesdeelíndicededeficienciainmunológicalacualseconsideroseruníndiceimportanteenlaseleccióndeagentesantitumorales.ElestudioinvestigoelefectodelFRC001sobrelainmunidadinespecíficaderatonesyGVHR.

MATERIALESYMÉTODOS1. Materiales

1.1. Medicamento:FRC001.

1.2. AnimalesExperimentales:Ratones,especieKumming,18~22gm,mitadmachos,ymitadhembras,proporcionadosporelExperimentalAnimalCenterofJiangsuTumorProphylactico-therapeuticResearchInstitute.

2. Método2.1. Funciónfagocíticademacrófagosmononucleares(pruebadeeliminacióndelcarbón)

2.1.1. Principiosexperimentales

RatonesexperimentalesfueroninyectadoscontintaIndiaelcualseusocomouncuerpoextrañogranular.LatintaIndiafuefagocitadayeliminadapormacrófagosmononuclearesdespuésdeentrarencirculación.El90%fuefagocitadoporlascélulashepáticasdeKupffermientrasqueelrestantefuefagocitadoporlosmacrófagosdelbazo.Latasadeeliminacióndecuerposextrañosenlacirculaciónreflejalafunciónfagocíticadelmacrófagomononuclear.Dentrodeciertosalcances,latasadeeliminacióndelcuerpoextrañogranulartuvounarelaciónexponencialconrespectoalaconcentracióngranularencirculación.Tieneunarelaciónlinealenunsistemadecoordinadasenlascualestiempoeslahorizontalylaconcentracióngranularellavertical.LainclinaciónKdelalíneaeslatasadefagocitosis(tasadeeliminación)

2.1.2. Manipulación

LosratonesfueronseparadosalazarenungrupodecontrolnegativoyotrogrupodecontrolpositivoygruposdedistintasdosisdeFRC001.Fueronalimentadosconaguadestilada,lecheelementalydistintasdosisdeFRC001por10días.CadaratónrecibióunainyeccióndetintaIndia(diluida1~5veces)0.05ml/1.0Gmdepesoporlavenadelacola30minutosdespuésdecomer.Enelprimerminuto(t1)yel5tominuto(t5)despuésdelainyección,seextrajo20mldesangredelavenapostorbitalconuntubodesucciónhumedecidoconsoluciónheparinaydiluidoen2mldesolucióndebicarbonatodesodioal0.1%.Semidiólaabsorcióndelasoluciónsanguíneaalos680nm.ParacalcularelvalordeKseutilizolasiguienteformula(lGAl-lGA5)/(t5-t1)=(lGA1/A5)/4

2.2. PruebaGHVR.

DosratonesdelíneapuraC57BLJCRhibridizadosparadaraluzaratonesreciénnacidosyelpadreexperimentalfueronalimentadosconFRC001por10díasmientrasqueelpadrecontrolnorecibiótratamientoalguno.Eneldía11devidalospadresfueronsacrificadosparaextraerlosbazos.Lasuspensión(1X108/ml)sepreparocontécnicaaséptica.Losratonesreciénnacidosfuerondivididosengruposexperimental,controlynormal.Losratonesreciénnacidosdelgrupoexperimentalfueroninoculadoscon0.1mldeunasuspensióndecélulasdebazodelpadre,losratonesreciénnacidosdelgrupocontrolfueroninoculadoscon0.1mldeunasuspensióndecélulasdebazodelpadre,yelgruponormalnorecibiótratamientoalguno.Todosestosratonesreciénnacidosfueronsacrificadosal7modíadespuésdelainoculación,ysusbazosfueronpesadosparacalcularelcoeficientede

bazo(mg.debazo/10mgdepeso).Luegosecalculóelíndicedeestimulacióndelbazo(SI)porlasiguienteformula:

SI=Coeficiente del promedio del bazo del experimental o control/promedio del coeficiente del bazo del grupo normal.

2.3. ElíndicedelbazoPruebadelíndicedelbazoydeltimo.RatonesdelaespecieKunming(18~22Gm)sedividieronalazarenungrupocontrol(alimentadoconN>S)grupodecontrolpositivoyelgrupoFRC001.Fueronalimentadosconelalimentorespectivopordiezdíasconsecutivosysacrificadospordesangradoorbitalalsegundodíadelaultimacomida.Susbazosytimosfueronextraídosypesadosexactamenteconunabalanzadetorsión.Losresultadosrevelaronelíndicedelórgano(bazootimo)/peso(mg/10mgdepesocorporal).

2.4. Pruebadetransformaciónlinfocitariadelratón(MTT)ColorimetríadelMTTdelbazodelratón.EspeciespurasdelratónKunmingfueronsacrificadospordesangradoorbitalylosbazosextraídosparahacerunasuspensióncelular5X10(8)/mldesuspensióncelularconlasolucióndeHankysuspensióndecélulasmedianasRPC1640.Sepusieron100mlalporodeunplatodecultivoyacadaporoseleagrego100mldePHA;mientrastantosecolocoelporodecontrol.Fueroncultivadosa37-38.5°C.enunincubadorCO2por72horas.Acadaporoseleagrego50mldeMTT4-5horasantesdeterminarelcultivo,luegocontinuarconloscultivos24,36y48horasrespectivamente.Sepusieronenunrefrigeradora4°Cporunanoche.Seretiraron150m.delsupernatantealqueseleagregó150mldeisopropanolymezcladohastatenerunasoluciónhomogénea.Laabsorción(A)decadatubofuemedidoa630nmdelfotómetroELISA.Paradetallesvealareferencia10.

2.5. PruebadehemólisisdelsueroRatonesdelaespecieKunmingde18~22gmfuerondivididosalazarengruposno-inmunológicos,inmunológicos;ygruposinmunológicoscondroga(dosisbaja,mediayalta).Acadaratóndelosúltimosdosgruposselesinyectointraperitonealmente0.2mldeglóbulosrojosdeoveja(SRBC)locuallodiluyóa3:5(V/V)conRatonesNSdelGrupodedrogaaloscualesselesdioladrogaantesodespuésdelainmunización.Cuatrodíasdespuésdelainmunización,sesaco1mlparaobtenerelsuero.Paramasdetallesvealareferencia8.

RESULTADOSYDISCUSIÓN1.ElefectodelFRC001sobrelafunciónfagocitariadeelmacrófagomononuclearinRatones

Figura1.-LacurvadelefectodeFRC001sobrelaFunciónFagocitariadelMacrófagoMononuclearenratones.

Elaumentodelíndicedeeliminacióndelcarbón(K)reflejalasmejorasdelafunciónfagocitariadelmacrófagomononuclearylainmunidadinespecífica.DeacuerdoconlaFigura1dosisbajas,medianayaltasdelFRC001aumentaronelvalordeKsignificativamente(p<0.01).ElresultadomuestraqueelFRC001puedeaumentarlafunciónfagocitariadelmacrófagomononuclearylainmunidadinespecífica.2.ElefectodelFRC001sobreelíndicedelbazoyelíndicedeltimo.(Tabla1)

Tabla1ElefectodelFRC001sobreelíndicedelbazo(SI)yelíndicedeltimo(TI)Elbazoyeltimosonórganosinmunológicosimportantes.Ladegeneraciónyatrofiadeellosinfluyesobresufunciónnormal.DeacuerdoconlaTabla1,FRC001tuvounefectoinsignificantesobreSIyTI(p>0.05).elresultadoenseñoqueEdfrannnotuvoinfluencia

evidentesobrelosórganosinmunológicos.Losautorespiensanquesedebaalahoradelainyección.3.EfectodelFRC001sobrelatransformaciónlinfocíticadelbazoenelratón.(figura2)

Figura2.ElefectodelFRC001sobrelatransformaciónlinfocíticadelbazoenelratón.Latransformaciónlinfocíticadelbazoreflejalafuncióninmunológicacelular.Latransformaciónlinfocíticaaumentóconeltiempo.Laamplitudenaumentoentrelahora26yla48fuelamayor.Comparadoconelcontrol,latransformaciónlinfocíticaalas24,36y48horastuvounaumentosignificativo(P;0.01).ElresultadomostróqueelFRC001puedeaumentarlafuncióninmunológicacelular.4.ElefectodelFRC001sobrelahemolisinadesuero(Fig.3)

Figura3.LacurvadeefectodelFRC001sobrelaformacióndehemolisinaensuero.

Lahemolisina(IgM)reflejalainmunidadhumoral.Elaumentodetiempomediodelaabsorcióndehemolisina(HC50)muestraelaumentodelahemolisinayelaumentodelainmunidadhumoral.DeacuerdoalaFig.3elHC50aumentogradualmenteconelaumentoenFRC001.Ladiferenciaentrelosgruposexperimentalycontrolfuesignificativa(P;0.01).ElresultadomostróqueEdfrannaumentolainmunidadhumoral.5.EfectodelFRC001sobreelGVHRenratones(Tabla2)

Tabla2.ElefectodelFRC001sobreGVHRenratones.GVHRreflejalafuncióninmunológicacelularporelíndicedelareaccióndelhuéspedytambiénelíndicedeestimulación.DeacuerdoconlaTabla2,elíndicedereaccióndelhuéspedyelíndicedelestimulodelgrupoFRC001fueobviamentemayoralgruponormal(sintratamiento)yelgrupocontrol(alimentadosoloconaguadestilada)(P<0.05oP<0.01).LosresultadosmuestranqueEdfrannpuedeaumentarymejorarlafuncióninmunológicacelular.FRC001tienealgunainfluenciaenlainmunidadinespecíficadelosratonesexperimentales.LafunciónreguladoradelFRC001sobrelainmunidadpuedeestarrelacionadoasuscomponentes.SehareportadoquelasmedicinasChinasquetienenlafuncióndefortalecerelKi,calentamientodelasangreydelYang,etc.Contienenmuchoscomponentesbioactivosquepuedenmejorarlafuncióndelsistemareticuloendotelial,laformacióndeanticuerpos,inmunidadespecificaeinespecífica.Además,laeliminaciónderadicaleslibresporloscomponentesdelamedicinachinatieneunafuncióncolaboradoraparaprotegerlosórganosinmunológicos,regulanlainmunidad,favorecenfactoresinmunológicos,etc.FRC001tienecomponentesquepuedenfortalecerelKiylasangre,calentarlasangreyeliminarradicaleslibres.Loscomponentesquedesempeñanunpapelpararegularlainmunidadysuintensidaddeacciónysusmejoresefectosbiológicosdeberánserestudiadosmasenelfuturo.

-The Experimental Study on the Effect of

FReOOl (China No.1 Tian Xian Liquid ) on Nonspecific Immunity of Mice

Robert W. Bradford D.Se.! , Kexiang Ding2

1. Bradford Research Institute, CA,USA Professor of Capital University, Washington DC

2. FRC Free Radical Biology & Medical Research Center

ABSTRACT This paper investigated the effect of FReaa 1 on nonspecific irnmunity of mice and gran versus host reaction (GYI-IR) .The results shows that FReaa 1 had some effect on phagocytic function of mononuclear macrophage.spleen index and thymus index, splenic lymphocyte transformation , serum hemolysin formation, host spleen reaction coefficient and stimulate conflicient and stimulus index i.e FRcaa 1 call increase and cnchancc humoral immunity and cellular immunity of experimental basis for the usc of FReOD 1 in regulating immunity and preventing and curing tumor. Key Word fReOOl Nonspecific immunity graft versus host reacti<;m(GVHR)

BACKGROUND

Tumor (ormation is related to immunologic inadequacy. un recentt year .lhcre arc many report on immunologic inadequacy index which was regarded as an important indrx in the screening of antineoplastic The study investigated I be efTcct of FReaa I on nonspccifie immunity of mice and

GVI-lR.

MATERIALS AND METHODS

I Materials

1.1 Subject:FRCOa I.providcd by U/rA'&7! Cb. LTj),J

1.2 Experimental Animal: Mousc,Kunming species, 18-22g, half male and half female .provided by Experimental Animal Center of Jiangsu Tumor Prophtlactico-therapeutic Reseach Institute.

2 Method: . 2.1 Phagocytic function of mononuclear macrophage(mouse carbon clearance test) 2 I. J Experimental principle:

Experimental mouse was injected with Indian ink which was used asgranular foreign body .The Indiau ink was phagocytized and cleared by mononuclear macrophage after it went into circulation.Ninety percent of it was phagocytized by liver Kupffer cell while the rest was

-phagocytized by spleen macrophage.The clearance rate of granular foreign body in ciculation reflected the phagocytic function of mononuclear macrophage. Within given scope.the clearance rate of granular foreign body had exponential function relation with granular dose.i .e. phagocytosis rate had direct propotion relation with granular concentration in circulation.They have linear relation in system of coordinates with the time as the abscissa and granular concertration as the vertical ordinate The slope K of the line is the phagocytosis rateCor clearance rate) .

2.1.2Man ipu lation Mice were randomly divided into negative control group,postive control grope and different

dose of PRCOO I groups.They were fed with distillcs water.elemene milk and different dose of FReaOI for succussivc 10 days.Each mouse was injected Indian ink(diluted to 1-5 times)O 05ml/l lag bw from mouse tail vein in 3a minutes after the last feeding.In the first minu te(t I )and Slh Illlllute( t5) after injection,20 J.1 I blood was drawn from postobital vein with suction

lube in which was moistened with heparin solution and diluted in 2ml 0.1% sodium carbonate solution .Then absorbance (A) of blood solution was assayed at 68anm .To calculate the value of K

by following Iomular (lgA l-lgAS)/ (t5-t l)=(IgA IIAS)I 4

2.2GVHH. test Two pure line mice t C5713L if- JCR hybridized to give birth to newborn mouse.Beginning at

the firs! day of the birth of the newborn mouse, experimental male parent were fed with fReaa I for 10 days while control male parent were not given any treatment.On the 11 th day after the birth of newborn mouse.male parent mice were killed by dislocation to take out spleens .Spleen cell suspension (l X was prepared by aseptic technique.The newborn mice were divided into

experimental group,controJ group and normal group .The experimental newborn mice were inoculated a Irnl spleen cell suspention of experimental male parent,the control newborn mice were jnoculatcdO lml spleen cell suspension of control male parent,and the normal group were not given any treatment .All these newborn mice were killed at seventh day after inoculation.and their spleens were weighed to calculate spleen coefficient (mg spleenfIOg.bw).Then calculation spleen stmulus index (SI) by following formula'

S I""'(Avcragc Spleen Coefficient of Experimental or Control )/ Average Spleen Coefficient of Normal Group

. 2.3 Spleen index and thymus index test

Kunming species mice (18-22g) were randomly divided into negative control group (fed with

N>S?) positive control group and FReaD 1 group. They were fed with resepective subject for successive 10 days.and killed by orbital bleeding on the second day after the last feeding.Their spleen and thymuses were stripped out and weighed accurately with torsion balance .The results

2

...

showed with organ index-weight of spleen or thymus(mg)/l Og.bw

2.4 Mouse lymphocyte transformation tcst-co;orimctry of mouse splenic MTT Pure species kUI1111ing mouse was killed by orbital bleeding and its spleen was stripped out to make 5 x IO(l)hnl cell suspension with Hank 's solution and RPM 1640 medium cell suspension 100 /1 I was added to pore of culture dish.and every pore was added PHA 100 f1 l and experimental drugs.in the meantime ,control pore was set up.They were cultured in °C .5% C02 incubator for 72hrs.Evcry pore was added 50 f1 I MTT 4-5hrs before the ending of culturc,then continue to culture 24h,36h,4Sh,respectively.They were put into 4 DC refrigerator for one night. Supernate 150 Ii I was sucked, added 150 f1 I acid isopropanol and blown to homogeneous solution. Absorbance (A) of every tube was assayed at 630nm ofELISA photometer. To see detail in reference] 10J.

2 5 Serum hemolysin test I Mice (Kuruniug species.l were randomly divided into nonimmunologic

group .immunologic group immunology and drug(low,middle and high dose) group.Every mouse of the last two groups was injected introper itoneally O.2ml sheep red blood cell(SRBC)which diluted to 3:5(V/V) with N.S Mice of drug group were given drug before or after immunization, Four days after irnrnuization. 1 ml blood was taken to get serum, To see the detail in referencc[8].

Result and Discussion

I. The effect of FRCOOI on phagocytic function of mononuclearmacrophage in mice (Figure 1)

0.018 (J Po> 0- 0,016 o ::l

n 0.0]4 (l> Po>

0.012 n (l>

....... ().(J\

0-(b x O.()OS --. .A

0.006

U.004

(),{)02

1 Co I. J v

Figure 1 The effect curve of FReOOl on Phagocytic Function of Mononuclearmacrophage in

3

- The increase of carbon clearance index (K) reflects the enhancement of phagocytic function of mononuclearmacrophage and nonspecific immunity.According to Figure I, low.middle and high dose of FReOOl increased K value significantly(p<O.OI).The result showed FReOOI can

enhance the phagocytic function of mononuclearmacrophage and nonspecific immunity, 2 The effect or fReoo I 0/1 spleen index and thymus index (Table J)

Table ¥! ._.._... effect of.......FRC001 on se.leen index thl:m_us index{TI) Group Number (n) Dose X±SD(mg/IOg.bw)

SI 1'1 , - •..._- _._ _-_ _ - - ----- ,- - - - - - - - ---'-- - - -_ ._- -_. ----

Control 10 a 69,59±25.59 26.23 ±9 .01

rncooi 10 G.O x lOung/kg x d) 64.92±35 .91 * 27.03±J3 ,03 * Elcmcne Milk '._I ]0 J00 X ] O(mg/kg x d) 78 ,26±22.82 * IG.15 ±2 .60 * * *

Compared wnh control, * P>O.OS ·, ** * * * P<O .OJ

Spleeu and thymus are important immunologic organs,The degeneration and atrophy of them will influence their normal [unction .According to Table I ,FReOOJ had no significant effect on SI and TI ([»0.05) The result showed Edfrann had no evident influence on immunologic organs. The authors thought it may be related to the shot time, 3.The effect of FRC001 on splenic Iyphocyte transformation in mouse(Figure 2)

Ab :;O I'b'HlCC

24 36 48 Time hour(h)

(Figure 2) The effect of FReaOl on splenic Iyphocyte transformation in mouse

Splenic lymphocyte transformation reflects cellular immunologic function .From Figure 2, FReaa I had evident influence on splenic lymphocyte transformation. Splenic lymphocyte transformation went up as the increasing of time.The increasing amplitude between 26h and 48h was the biggest Compared with control.splenic lymphocyte transformation at 24h,36h,and 48h increased signiticantly(P<O,aI),The result showed FReOOJ can enhance cellular immunologic function .

(,I ) 3 2.5

2 1.5

I 0.5 o

mTimc Hour Tesl

IIEI TilllC Hour Control

4

4.The effect of FRC001 on serum hemolysin (Figure 3)

.--- - - - - --_ ._-- -. ._ - - - - - - - - - - - - - - -..,

Hemolysis (!lUll )

....

- _ ..._.....- . ._ -----_._ - ----- - - -------'

(Figure 3) The elTcct Curve of FRCOOl on serum hemolysin Formation.

Hemolysin(lgM) reflects humoral immunity. Increasing of absorbance at the time half hemolysin takes plaee(HC50) shows the increasing of hemolysin and enhancement of humoral immunity .According to Figure 3,HC50 went up gradually as the increasing of fRCOO 1.The difference between experiment group and control group was significant (P<O .Ol).The result showed Edfrann had enhance humoral immunity.

5.The effect of FRC001 on GVHR in mice (Table 2)

.. ...... ... ......I!!£ effect of FReaOl on GVHR ill mice Group Number Administration Dose Host Reaction Stirnulu Index

.. .. .. ..._._. __ .. .. .. (mg/kg X d) Index(mg spleen/g.w) (Sl) Normal 8 19 o 7.76±1.14*** Control 8 19 6.0 X 10 9.82±1.36** 1.22 PRCOOI 8 19 6.0 X JO 12.96±1.88 196 ..._-_.£-........._......... ...__.....,......._...

Compared with FRCOO I group ,*P>O.05; **P<O.05; "'* *P<O.O 1

Gy}-m reflects cellular immunologic function by host reaction index and stimulus index. According to Table e, host reacyion index and stimulus index of FReOO I group was obviously higher than normal group(witbout any treatment) and control group (fed with distilled water only)(P<O.05 or P<O .O I).Thc result shows Edfrann can increase and enhance cellular immunologic function.

5

-

All of these results shows FRCaa 1 had some influence on nonspecific immunity of experimental mice,The regulation function of FRcaa I to immunity may be related to its componets.It is reported that Chinese drugs with the function afinvigprating Qi,b/ood and warming Yang,etc contain many bioactive components which can enhance the function of reticuloendothelial system.antibody formation,specific and nonspecific immunity.In addition,free radical scavenges in Chinese drugs have cooperative function in protecting immulogic organs,regulating immunity.enhancing immunologic factors.etc . FReaOl contains components which can invigorate Qi and blood, warm blood and scavenge free radicals .then the cornponens which play roles in regulating immunity and their action intensity.best bioeffect are waiting for future study.

REFERENCES [I] Yu He, eL al Tumor and Immunity, Shanghai Science and Technology Press, 1982, 114

[2J Cai Limian .Taug Zhangyou, Study on Growth Condition of Primary Carcinoma of Liver and Host Macrophage Activity, 1989,8(4):264

[3] Yang Guizhcng, Xu Yiping, Lymphocyte function Test, Clinical Immunologic Tcchonology.Shaughai Science and Technology Press, 1982 :314

l4J Wang Yixizn, et al . The Study on Antitum Science and Technology Press ,Bijing.l996)3

[5J. Zhou Junfang, ct al. The Effect of Stress on Tumor Metastasis and the Immunologic Mechanism, Cancer 1992,

[6] Cheng Qin, Antiaging Research Experimentnal Methods, Chinese Medical Science and Technology Press , Beijing, 1996,

17J- Tao Yixun, ct al Clinical Immunologic Test, Shanghai Science and Technology Press , 1983 : 105 [8] Xu Xueying, et al An Improved Humoral Immunity Test-Hemolysin Assay.Pharmaceutical Acta

1979,14(7):443

[9] Xu Shikai et al .Antiaging Materia Medics, 1'1 edition Chinese Medical Science and Tcchonology Prcss.Bijing , 1995, 164- 165

[I 0J,Li Dcnghua, Tian Zhigan, Zhang lie, et ai, Rapid DetermInation of Cytotoxicity of Drugs on Supeudcd Tumor Cell, 1991, 10(3):226-228

[I 1J Ader R. Psyvhoncu 1'0 immunology .New York, Academic Press Inc, 1981: 37

[12J Tsutornu Okimura, et al Effect of Restraintatress on Delay Typer-sens-activity and Phagocytosis in Mice.Jean.J Pharmacol , 1986,41:229

[13] Brown RE et al Fatty Aids and Inhibition of Mitogen-Induced Lymphocyte Transformation by leukemic gerum. J Imruunol, 1983 ,131 : 10 I

(j