HBsAg进入体外培养的人绒毛膜滋养层细胞的初步研究

目的：检测HBsAg进入体外培养的人绒毛膜滋养层细胞的情况，为HBV宫内传播机制研究提供理论依据。方法：用胰蛋白酶（0．25％）－DNase I(0.15U/ml)联合消化法，获取纯度较高的人绒毛膜滋养层细胞。将状态良好的对数生长期人绒毛膜滋养层细胞分为6组，HBsAg-anti-HBs复合物组（I），热灭活的HBsAg、HBsAg、anti-HBc均阳性血清和高滴度anti-HBs阳性血清共同孵育物组（Ⅱ），热灭活的HBsAg、HBeAg、anti-HBc均阳性血清组（Ⅲ），HBsAg组（Ⅳ），热灭活的正常人血清组（Ⅴ），及正常细胞培养液组（Ⅵ，空白对照组）。分别收集各组细胞铺片，免疫组化检测细胞的感染状况。结果：所获人绒毛膜滋养层细胞纯度较高，符合后续实验要求。用鼠抗人anti-HBs免疫组化检测收集的细胞铺片，发现I、Ⅱ组均有大量HBsAg阳性信号，Ⅲ组细胞亦有少量HBsAg阳性信号出现，其他各组细胞均未发现HBsAg阳性信号。结论：体外培养的人胎盘滋养层细胞能将HBsAg-抗HBs复合物中的HBsAg，但不能将单独的HBsAg“摄入”胞内。

HBsAg uptake into human trophoblasts cultured in vitro]

OBJECTIVE: To detect the entry of HBsAg into human trophoblasts cultured in vitro, to provide some clues for the mechanism responsible for HBV intrauterine transmission. METHODS: Digestion with both trypsin (0.25%) and Dnase I(0.15 U/ml) followed by repeated purification was performed to obtain a homogeneous population of trophoblast cells. The well-developed cells during longitudinal phase were randomly divided in 6 groups, which were co-incubated respectively with HBsAg-anti-HBs complex (I), co-culture of heat-inactivated sera positive for HBsAg, HBeAg, and anti-HBc and sera with high titer anti-HBs(II), heat-inactivated sera positive for HBsAg, HBeAg, and anti-HBc(III), HBsAg (IV), heat-inactivated normal sera (V), and normal medium(VI). The coverglasses mounted with confluent cells were harvested for anti-HBs immunohistochemical staining. RESULTS: The isolated cells comprised a highly purified villous trophoblast population and could meet the demands of further experiments. After co-incubation of the cells with different agents, HBsAg test yielded positive results in the cells co-incubated with the HBsAg-anti-HBs complex and in the co-cultures of heat-inactivated sera positive for HBsAg, HBeAg and anti-HBc with sera containing high-titer anti-HBs. HBsAg test was negative in all other cell groups. CONCLUSION: Human trophoblast cells could take in HBsAg in the form of HBsAg-anti-HBs complex instead of single HBsAg molecule.