HBsAg进入体外培养的人绒毛膜滋养层细胞的初步研究

HBsAg uptake into human trophoblasts cultured in vitro

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(0.15 U/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、HBeAg、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。

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 DNaseⅠ(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.