人胎盘滋养层细胞的分离培养及HCV体外感染试验

目的：体外分离培养较高纯度的胎盘滋养层细胞，观察其生物学特性，探讨丙型肝炎病毒（HCV）经胎盘母婴传播的具体途径。方法：采用胰蛋白酶消化法消化人足月胎盘组织，以35％，45％两个Percoll密度梯度进行分离纯化。并用免疫组化及透射电镜等技术对其生物学特性进行观察。HCV阳性血清感染体外分离培养的胎盘滋养层细胞，通过逆转录聚合酶链反应及扩增敏感试验对培养上清进行定性、定量检测，以观察HCV的感染及复制情况。结果：胎盘组织中滋养层细胞角蛋白染色阳性，血管内皮细胞及基质成分波形蛋白染色阳性，经该法分离纯化的细胞角蛋白染色阳性者（滋养层细胞）占90％以上，电镜下滋养层细胞可观察到成熟型、中间型、未成熟型三种形态。HCV感染细胞培养16d内培养上清中可间断检测出HCV RNA.结论：改进后的分离方法可以得到较高纯度的滋养层细胞，HCV感染胎盘滋养层细胞可能是HCV宫内母婴传播的途径之一。

Isolation and characteristic of villous cytotrophoblast form term human placenta and hepatitis C virus infection of human term syncytiotrophoblast cells cultured in vitro

AIM To isolate cytotrophoblasts from human placentae and investigate the specific route of mother to infant transmission of hepatitis C virus (HCV). METHODS Cytotrophoblasts were prepared from human term placentae digested with trypsin, and centrifuged on two layers (35% and 45%) of Percoll gradient. Biological characteristics were observed by the way of immunohistochemistry and transmission electron microscope. Syncytiotrophblasts were incubated in HCV positive serum for 24 hours, and then the HCV infection and replication of syncytiotroblasts were observed with RT PCR. RESULTS In human placentae, the trophoblast layer was positive to cytokeratin and negative to vimentin, while the endothelial cells of blood vessel and hypothallus were negative for cytokeratin and positive for vimentin. More than 90% of cytokeratin positive cells (trophoblast) were obtained through our improved method. Under transmission electron microscopy, we have observed three different mature stages of cytotrophoblast. The HCV RNA was intermittently detected in the supernatent of the culture medium during the 16 days culture. CONCLUSION Higher purity trophoblasts can be obtained by this improved method. HCV infection of syncytiotroblasts is probably one of the routes of HCV mother to infant transmission in uterine.