HBV在原代胎盘滋养细胞内复制的初步探讨

发布时间：2018-04-02 15:31

本文选题：乙型肝炎病毒　切入点：滋养细胞　出处：《现代妇产科进展》2015年01期

【摘要】：目的:从体外实验研究HBV在胎盘滋养细胞的感染和复制,初步探讨HBV经胎盘传播的机制。方法:收集HBV携带孕妇足月胎盘12例,采用密度梯度离心法联合反复贴壁法分离、纯化滋养细胞,提取滋养细胞进行培养。流式细胞术检测滋养细胞中CK-7表达,进行细胞纯度鉴定;免疫双标法结合激光扫描共聚焦显微镜技术检测滋养细胞CK-7及间质Vimentin的表达,进行细胞形态学鉴定。PCR-荧光探针法检测体外培养24、48、72h滋养细胞上清HBV DNA的表达;微粒子酶免疫分析技术检测体外培养24、48、72h滋养细胞上清HBs Ag和HBeAg的表达。流式细胞分选技术分选出纯度98%以上的滋养细胞,荧光定量PCR检测滋养细胞中HBV ccc DNA的表达。结果:12例HBV携带孕妇胎盘滋养细胞体外培养24、48、72h,上清均可检测到HBV DNA和HBsAg、HBeAg的阳性表达。HBV-M滴度在24h表达量最高,随着时间的推移,HBV DNA滴度逐渐下降,而HBs Ag和HBe Ag滴度呈先下降后上升的趋势,但3个时间点均无统计学差异(P0.05);3个时间点上清HBs Ag和HBe Ag的表达量有良好的相关性(P0.05)。12例HBV胎盘滋养细胞标本中,4例检测出HBV cccDNA阳性,阳性率为33.3%,表达量(copies/ml)分别为5.67×103、2.12×104、1.72×104、4.90×104。Pearson相关分析结果显示,母血HBV DNA滴度和滋养细胞HBV cccDNA滴度呈良好的正相关(r=0.977,P=0.023)。结论:HBV可感染胎盘滋养细胞并在滋养细胞内繁殖复制。
[Abstract]:Aim: to investigate the infection and replication of HBV in placental trophoblast in vitro and to explore the mechanism of HBV transmission through placenta.Methods: twelve pregnant women with HBV carrying placenta were collected and separated by density gradient centrifugation combined with repeated adherent method. Trophoblast cells were purified and cultured.The expression of CK-7 in trophoblast cells was detected by flow cytometry, and the expression of CK-7 and interstitial Vimentin in trophoblast cells was detected by double immunostaining combined with laser scanning confocal microscopy.The expression of HBV DNA in the supernatant of trophoblastic cells cultured in vitro was detected by microparticle enzyme immunoassay, and the expression of HBs Ag and HBeAg in the supernatant of cultured trophoblastic cells was detected by microparticle enzyme immunoassay.More than 98% of the trophoblastic cells were isolated by flow cytometry, and the expression of HBV ccc DNA in trophoblast cells was detected by fluorescence quantitative PCR.Results the positive expression of HBV DNA and HBV-M was detected in the supernatant of 12 cases of HBV carrying pregnant women placental trophoblastic cells for 24 h and 48 h for 72 h. The titer of HBV-M was the highest in 24 h, and the titer of HBV-M decreased gradually with the passage of time.However, the titers of HBs Ag and HBe Ag decreased first and then increased, but there was no significant difference among the three time points, and the expression of HBs Ag and HBe Ag in the supernatant at 3 time points was positively correlated with that of HBV placental trophoblastic cells in 4 out of 12 HBV placental trophoblastic specimens.The positive rate was 33.3.The positive correlation analysis showed that the positive correlation between maternal HBV DNA titer and trophoblastic HBV cccDNA titer was 0.977P0.0233.The positive correlation was 5.67 脳 10 3 / ml (2.12 脳 10 4) and 1.72 脳 10 4 (4.90 脳 10 4) 脳 104.Pearson, respectively.Conclusion\\\% HBV can infect placental trophoblast and reproduce in trophoblast.
【作者单位】：广州医科大学附属第三医院妇产科;暨南大学附属第一医院妇产科;陕西省人民医院妇产科;广东省计划生育科学技术研究所;
【基金】：国家自然科学基金项目(No:C30973214) 高等学校博士学科点专项科研基金(No:20070559006)
【分类号】：R512.62

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