乙型肝炎病毒特异性转移因子的研究.docx
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乙型肝炎病毒特异性转移因子的研究.docx
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乙型肝炎病毒特异性转移因子的研究
乙型肝炎病毒特异性转移因子的研究
来源:
青年人(Qnr.Cn) 更新时间:
2010/3/1117:
24:
21 【字体:
小大】
摘 要 制备一种乙型肝炎病毒特异性转移因子制剂(HBV-STF),为临床应用提供有价值的实验依据。
从人HBsAg阳性胎盘中制备了HBV-STF,并对其理化性质、免疫学活性进行了检测和初步的临床试用。
每批样品经无菌试验、热原质检查、动物安全性试验等均符合药典要求。
本品最大紫外吸收光谱在256±2nm处,E260nm/E280nm比值大于2.7。
其水解氨基酸含17种。
对人T淋巴细胞E受体的激活试验结果显示,HBV-STF的Ea-RFc平均增高率在83.47%~103.48%之间,抗原特异性皮肤试验表明HBV-STF能刺激小鼠体内T淋巴细胞增殖,诱导小鼠跖趾部皮肤的迟发性变态反应。
对HBV-STF的初步临床试用也取得明显效果,显示HBV-STF是一种可用于治疗乙肝的安全、有效的免疫调节剂。
关键词 乙型肝炎病毒特异性转移因子;制备;理化性质;免疫学活性
StudiesofHepatitisBVirus-SpecificTransferFactorPreparation
LinYuanzao,ZhaoAiping,ChenGengfu,ChenWeiqiang,
HuangQingsong,LuoLiewei,LiHongzhi,LaiMuxian
(DepartmentofBiotechnology,GuangdongCollegeofPharmacy,Guangzhou510224,China)
Abstract ToestablishanewmethodforthepreparationofhepatitisBvirus-specifictransferfactorpreparation(HBV-STF),andhencesomeavailabledataforcomparativestudyofclinicalapplicationaredocumented.HBV-STFwaspreparedwithhumanplacentalfromHBsAgfemalepatientspositive.ThephysicalandchemicalqualityanditsimmunologicalpropertiesofHBV-STFpreparationweretested.Alsopreliminaryclinicaltrialwasused,demonstratesaconsistencyinqualityamongeachbatch.Resultsfrombacteriaculture,safetyandpyrogentestsandacutetoxicityinmiceallsatisfiedwiththerequirementsofthepharmacopoeia,indicatingthatthepreparationsweresafe.Themaximumabsorptionofultravioletspectrawasat256±2nm,TheE260nm/E280nmisgreaterthan2.7,whichisbetterthantheinternationallyusedcriterion,i.e.E260nm/E280nm≥1.8.Thepreparationscontained17kindsofaminoacids.EffectofHBV-STFonactivationofEreceptorsofhumanTlymphocytesshowstheincreasingraterangingfrom83.47%to103.48%,HBV-STFcanstimulatemultiplicationofTlympholeukcyteandinducingdelayedallergyinmousemetatarsophalangealskinbyantigenspecificskintest,indicatingthepreparationshavestrongimmunologicalactivities.Preliminaryresultsfromclinicaltrialalsohaveobviouscurativeeffect.TheresultsshowedthattheHBV-STFpreparationwasasafeandhigheffectiveimmuno-regulatorfortreatingviralhepatitisB.
KeyWords HepatitisBvirus-specifictransferfactor(HBV-STF),Preparation,Physicalandchemicalquality,Immunologicalactivities
CLCnumber:
R978 DocumentCode:
A
ArticleID:
1005-8915(2000)03-0141-05
Thetransferfactorpreparation(TF)isanimmunologicalactivemixturewhichispresentinsidewhitebloodcellsanddialysate.Thespecifictransfer-factorpreparation(STF)isamixturewhichdependsonitsantigenforthebiologicalactivities[1,2].ThenatureofSTFprovidesuswithanewwayforpreparation.OurlaboratoryextractedHBV-STFfromHBsAgpositivehumanplacenta,detectedsomeofthepropertiesofHBV-STFandconductedthepreliminaryclinicaltrialofthepreparation.Theresultsarereportedhere.
1 MaterialsandMajorequipment
1.1 Materials
HBsAgpositivehumanplacentawereprovidedbytheDepartmentofObstetricsandGynecologyatHaizhuWomenandChildrenhospital.Dialysistubeswithmolecularweightcuttingoff10kDwerefromFrance.Hybridmice(maleandfemale,17~28g)wereprovidedbydepartmentofanimalofourcollege,NewZealandwhiterabbits(maleandfemale,2.5~3.4kg)wereprovidedbytheBiologicalInstituteofGuangdong.
1.2 Majorequipment
MDF-292ultra-lowfreezer(-85℃,Sanyo,Japan);ZK380ultracentrifuge(RertholdHermleGbHoeCo.,Germany);PCRthermalcycler(EPOCHCo.,Tianjin);Aminoacidanalyzer(Walters,USA);752UV-spectrophotometerand722Visible-lightspectrophotometer(ThethirdfactoryforanalysisinstrumentsofShanghai).
2 Methods
2.1 Preparationoftransferfactor
Afterfatandconnectivetissueswereremoved,HBsAgpositiveplacentawerehomogenizedathighspeedwithequalamountof0.9%NaCl.Thehomogenatewerestoredat-85℃overnightandthawedat37℃.Afterseveralcyclesoffreezingandthawing,thesupernatantswerecollectedwithultracentrifugationandsterilizedviadialysisandfiltration.Thebacteriaculture,pyrogentest,animaltoxicity,HBV-PCRandHBsAgtestwereperformedtoensurethesafetyofthepreparation.Thepreparationwaspackagedat2ml/Ampule.
2.2 Polypeptidecontentsmensuration
ItwasmeasuredbyLowrymethod.
2.3 RNAcontentmensuration
Itwasmeasuredbyorcinoltest.
2.4 HBV-PCRdetection
PCRanalysiswereperformedaccordingtothemanufacturer'sinstruction.ThePCRproductswereelectrophoresizedin2%agarosegelandcomparedwiththepositivecontrolat314bpposition.
2.5 Immunologicalactivityassays
TheimmunologicalactivityassayswereformedwithEa-RFcmethod[3].Briefly,pancreatin(Difco,Detroit,MI,USA)wasdissolvedinHank?
ssolution(1%)andstoredat-20℃.Thecontroltubewasaddedwith0.2mlsupernatantandtissueculturemediumRPMI1640withoutTF.Theexperimentalgroupswereaddedwith0.1mlHBV-STF,P-TFandRPMI1640medium.Thetotalvolumeineachtubewas1ml.Afterincubatedat37℃for18hours,eachtubewaswashedwith5timesofice-coldHank?
ssolutiontwice.Thepelletswereresuspendedin0.2mlRPMI1640forEa-RFcassays[4].Theresultswerecalculatedasfollowing:
2.6 Antigenspecificskintest
ThirtyhealthyNIHmiceofabout20geachinweight(providedbytheExperimentalAnimalCenterofZhongshanMedicalUniversity)weredividedinto3groups.ThesubcutaneousinfectionofHBV-STF0.5mlwasgivenintotheabdomenofeachmouseinthetestergroup,onceeveryotherday7timesaltogether.Aweekafterthelastinjection,thefeetofthehindlegsonbothsidesofthemicewereinjectedwith1∶25hepatitisBvirusvaccine0.5ml(producedbyShenzhenKangtaiBiologicalProductscompanyLtd.)72hoursaftertheinjection,thehindlegsonbothsidesofallthemicewerecutoffrespectively.Pathologicalsectionwasthendoneofthemetatarsusskintakenfromthehindlegsafterformaldehydefixation,forthepurposeoftheobservationoftheinfiltrationofthelymphoecyteindermaandsubcutaneoustissues.Thecontrolgroup,withthesubstitutesofP-TF(producedbyGuangdongBiologicalProductsInstitute)andphysiologicalsalineforHBV-STF,wasemployedforintegrationcontrol.
3 ResultsandDiscussion
3.1 HBV-STFqualitycontrolresults
Tab1demonstratesaconsistencyinqualityamongeachbatch.Resultsfrombacteriaculture,safetyandpyrogentestsandacutetoxicityinmiceallsatisfiedtherequirementsofthepharmacopoeia,indicatingthatthepreparationsweresafe.SincethepreparationswereextractedfromHBsAgpositiveplacenta,inactivationofHBVwascritical.Wangetal[6]incubatedasimilarpreparationat56℃for10hoursandtreateditwith0.25%formalin.WefoundthatalthoughHBsAgwasnegative.ThefigureandTable1showtheresultsoftherandomtestsofthreebatchesofpreparations.Theimmunologicalactivityofthepreparationinvitrowasalsonegative.WebelievethatTFisaheat-sensitiveandwillbedenaturedat56℃forjust45min.ThemethodreportedhereallowedustoremoveHBVandevenresidualHBV-DNAat4℃asdeterminedbyPCRandHBsAgdetectionassays.Moreimportantly,themethodswereabletopreservetheHBV-STFactivity.
Tab1 DetectionresultsofHBV-STF
Item
941223
950120
950309
Aspect
Δ
Δ
Δ
pH
6.9
6.8
6.9
Ultravioletspectra
E254nm
0.801
0.839
0.846
E260nm/E280nm
>2.70
>2.75
>2.90
Proteinreaction
-
-
-
Polypeptide(mg/ml)
0.37
0.39
0.41
RNA(mg/ml)
0.19
0.20
0.22
HBsAg
-
-
-
HBV-PCR
-
-
-
Asepsistest
Δ
Δ
Δ
Safetytest
Δ
Δ
Δ
Activitytest(%)
Δ
Δ
Δ
Totalevaluation
Δ
Δ
Δ
-Negativereaction,ΔPastmuster
Thefigure1indicatesthatthepreparationhadtwopeaksappearedfrom200nmto300nm,whichwassimilartootherTF.Themaximumabsorptionwasat256±2nm.Therewasaparallelabsorptionfrom254~260nmwithmoreoccurredat254nm.Thispositionwassimilartothatatwhichplacentalabsorptionoccurred,suggestingthatthepreparationofHBV-STFmaycontainplacentaSTF[6].TheE260nm/E280nmisgreaterthan2.7,whichisbetterthantheinternationallyusedcriterion,i.e.E260nm/E280nm>1.8.
Fig1 UltravioletspectraofHBV-STF
3.2 Contentsofaminoacids
Tab2illustratesthecontentsofaminoacids.Thepreparationscontained17kindsofaminoacids.
Tab2 ContentsofaminoacidsinHBV-STF
Aminoacid
Contents
(mg/dL)
Aminoacid
Contents
(mg/dL)
Asp
1.867
Tyr
1.202
Glu
10.666
Val
10.678
Ser
1.799
Met
2.516
Gly
8.790
Cys
0.221
His
3.587
Ile
4.620
Arg
0.189
Leu
16.044
Thr
4.082
Phe
7.496
Ala
13.088
Lys
5.102
Pro
16.544
Generalcontents
108.5
3.3 EffectofHBV-STFonactivationofEreceptorsofhumanTlymphocytes
Table3showstheresultsfromEa-RFcassays.ThesecondnationalmeetingonTFadoptedEa-RFcorEt-RFctestasastandardmethodtodeterminetheimmunologicalactivityofTFwithasatisfactoryvalueofgreaterthan20%increasingrate[8]Weobtainedtheincreasingraterangingfrom83.47%to103.48%,indicatingthepreparationshavestrongimmunologicalactivities.
Tab3 EffectofHBV-STFonactivationof
EreceptorsofhumanTlymphocytes
Group
N
Ea-RFc%
(
±s)
Incremental
averagevalue(%)
941223
5
21.1±2.3
83.47**
950309
5
23.4±3.6
103.48**
950401
5
22.3±4.5
93.91**
P-TF
5
15.2±2.8
32.26*
Control
5
11.5±3.2
*P<0.05,**P<0.01,comparedwithcontrol
3.4 ImmunologicalspecificityofHBV-STF
Themouse'sskinwasinjectedwithHBV-STF,P-TFandphysiologicalsalinerespectivelyforthedelayedallergy.Theinfiltratestatusofthelympholeukcyteinshinwasobserved.Thecriteriaforevaluationis1scoreforalittleandscatteredinfiltratelympholeukcyte,2scoreformoreandasystematicinfiltratelympholeukcyteorfocusconcent
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- 乙型肝炎 病毒 特异性 转移因子 研究
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