HBV感染胎盘滋养细胞的体外研究.doc

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1、HBV感染胎盘滋养细胞的体外研究作者：陈天艳，陈云茹，刘敏，牛迎花，蔺淑梅，叶峰，张曦，刘锦锋，赵英仁，张树林【摘要】　　目的：对早孕绒毛的滋养细胞进行原代培养，利用HBV感染血清对滋养细胞进行体外感染，探讨细胞在体外培养条件下对HBV的感染率及HBV在细胞中的复制状态.方法：利用胰蛋白酶和胶原酶联合消化培养法原代培养早孕胎盘中的滋养细胞，并进行免疫组化鉴定，获得纯度较高的滋养细胞后进行体外的HBV感染，通过细胞免疫化学染色、ELISA及PCR法分别检测细胞上清中的HBsAg，细胞中的HBVDNA及cccDNA.结果：原代培养的滋养细胞，细胞生长良好，特

2、异性角蛋白7阳性，纯度高.原代滋养细胞在与HBV阳性血清共同孵育后均未出现明显的细胞病变，感染后24h和48h在滋养层细胞细胞浆中可见HBsAg的阳性表达，被感染细胞呈散在分布.感染后48h起可检测到弱阳性的HBsAg及HBVDNA，感染后细胞内未检测出cccDNA.结论：利用联合酶消化法建立了胎盘中滋养细胞的体外培养系统.HBV能够在体外感染原代培养的滋养细胞，对被感染细胞的生长无明显影响.HBV可能不在滋养细胞中复制.【关键词】乙型肝炎病毒；滋养层细胞；原代细胞培养【Abstract】AIM:Tosetuptheprimaryculturemode

3、loftrophoblasticcellsfromhumanplacentaandtoinvestigatetheinfectionratioandreplicationoftrophoblasticcellscoculturedwithHBVinfectionserum.METHODS:Usingtrypsinandcollagenaseassociateddigestion,trophoblasticcellswereobtainedfromhumanplacenta.Immunohistochemistrystainingofthecellswe

4、redoneforidentificationofprimaryculturecellsbyspecificanticytokeratin7.TrophoblastcellswerecoculturedwithHBVinfectionserumfor24h.TheinfectionefficiencywasexaminedbyELISAofculturesupernatants,immunohistochemistryofcellslidesandPCRamplificationforHBVDNAandcccDNAofinfectedcells.RE

5、SULTS:Theprimaryculturemodeloftrophoblasticcellsfromhumanplacentawasestablishedsuccessfully.ObservationofthemorphologyoftrophoblasticcellsshowedthatnosignificantcytopathiceffectwasfoundwhencoculturedwithHBV.Nosignificantdestructionofcellularstructureorcellcelljunctionwasfound.C

6、ulturemediumwascollectedat24,48hoursafterinfection,respectively.SecretionofHBsAgwasobservedat48hourspostinfection.BothmembraneandcytoplasmiclocalizationofHBsAgwereobserved.Theinfectedcellswereinadiffuseddistribution.ThefrequencyofHBsAgpositivecellsatdifferenttimepointsdidnotshow

7、anysignificantchanges.HBVinfectedcellshadverylightspecificbandofHBVDNAandnocccDNAwasobserved.CONCLUSION:Weestablishaprimaryculturemodelof6trophoblastcells.OurstudyindicatesthattrophoblasticcellsculturedinvitrocanbeinfectedbyHBV.Buttheefficacyofinfectionislow.ThereplicationofHBVin

8、trophoblasticcellsisnotobserved.【Keyword