反义寡核苷酸对H1δ9细胞中了型肝炎病毒

在Ｈ-１δ９细胞中观察反义寡核苷酸（ＡＳＯＤＮ）及其硫代修饰物（Ｓ－ＡＳＯＤＮ）对丁型肝炎病毒（ＨＤＶ）基因复制与表达的抑制作用。方法在分子水平证实互补于ＨＤＶ基因链核酶自裂位点和Ｓｔｅｍｌ区的ＡＳＯＤＮ可抑制核酶自裂的基础上，进一步台成针对该区６８４－６９８位核苷酸的１５聚ＡＳＯＤＮ及Ｓ－ＡＳＯＤＮ，在培养的ＨＩδ细胞中加人不同寡核苷酸，分别采用ＥＬＩＳＡ和斑点杂交法检测上清中ＨＤＡｇ及细胞中的ＨＤＶｃＤＮＡ。结果加人终浓度为６μｍｏｌ／Ｌ的Ｓ－ＡＳＯＤＮ后２４ｈＨ１δ９细胞中ＨＤＶＲＮＡ复制及ＨＤＡｇ分泌均受抑制，抑制率分别为８４５％和７６．１４％。Ｓ－ＡＳＯＤＮ的终浓度为２、４、６μｍｏｌ／Ｌ时，其抑制作用呈剂量依赖关系。在同等剂量时，ＡＳＯＤＮ与Ｓ－ＡＳＯＤＮ抑制作用相近。结论提示所用ＡＳＯＤＮ及Ｓ－ＡＳＯＤＮ均能有效抑制转基因细胞中ＨＤＶ基因的复制与表达，为进一步在动物体内研究Ｓ－ＡＳＯＤＮ抗ＨＤＶ的作用提供了实验依据。

Inhibitory effects of antisense oligodeoxynucleotide on replication and expression of HDV genome in vitro and in vivo

Objective:To studytheinhibitoryeffectsof antisenseoligodeoxynucleotide(ASODN)anditsthiophosphate(S-ASODN)on thereplicationandexpressionof hepatitisD virus(HDV)in H1δ9cellsandin tupaiabody.Methods:After15mer-ASODNandS-ASODNweresynthesized,differentconcentrationsof ASODNandS-ASODNwereaddedto theculturemediumof H1δ9celllineandthenHDAgin thesupernatantof theculturewas examinedwithELISAand HDV-RNAinthecellsdeterminedwithdotblothybridization.SixteentupaiaeweresuccessfullyinfectedwithHDVand thenequallyrandomizedinto2groups.In thetreatedgroup,theanimalsreceivedintravenousinjectionsof S-ASODN3mg everyotherdayfor7timesandthecontrolgroupwereinjected withsamevolumeof normalsaline.On the5th,10th,15thand20thdayaftertheadministration,samplesof bloodandlivertissueswereexaminedwithimmunohistochemical methodforHDAganddotblothybridizationandin situ hybridizationforHDV-RNA.Results:Twenty-fourhoursafter theadditionof a finalconcentrationof6μmol/Lof S-ASODN,thereplicationof HDV-RNAandthereleaseof HDAgin theH1δ9cellsweresuppressedby84.5%and76.14%respectively.Theinhibitionwas dose-dependentwhenthefinal concentrationsof2,4and6μmol/Lof S-ASODNweregiven.WhenASODNandS-ASODNwereadministeredinthe samedosage,theinhibitionshowedno significantlydifferencebetweenthe2agents.On thelastdayof theadministration of S-ASODN,7outof the8tupaiaeof thetreatedgroupshowednegativeHDAgandHDV-RNAinthelivertissuewhile only1outof the8tupaiaeof thecontrolgroupwasnegative.Tendaysafterthecessationof drugadministration,3tupaiae of thetreatedgroupand7of controlwerepositiveof HDAgandHDV-RNA.Conclusion:OurfindingsshowthatS-ASODNefficientlyinhibitesthereplicationandexpressionof HDVgeneinH1δ9cellsandin thebodyof tupaia,which providesanexperimentalbasisfortheanti-HDVapplicationof antisenseoligonucleotides.