Establishment Of Human Trophoblasts Culture Method And Primary Studies On Entry Of HBsAg Into Human Trophoblasts

Aim HBV could infect fetus through placentae, which is intrauterine transmission of HBV. At present, there were only a few reports about the mechanism of HBV intrauterine transmission. Since 1992, our department have performed sysematical studies in this field and our studies suggested that HBV intrauterine transmission may occur either through placental tears with transfusion of infected maternal blood into the fetal circulation, which is the named "hematogenous transmission" , or through progressive infection of different placental layers until the virus reaches the fetoplacental circulation, which is the named "transplacental cellular traffic" . But much further work still is needed for the confirmation. Trophoblast cells, as the outermost covering of placental barrier, are directly bathed in the maternal blood, might be the key sites for invasion of HBV into placentae. The human trophoblast is known to serve several roles inpregnancy, including participation in numeral virus intrauterine transmission. However, the mechanism of entry of HBV into trophoblast cells has not been reported yet. In vivo studies indicated that trophoblast cells in HBsAg positive pregnant women were positive for both HBsAg-anti-HBs complex and IgG FcR. In the present paper, we were about to simulate HBV infection of cultured trophoblast cells in vitro. Methods HBV infection markers negative placentae (6-10 weeks gestation) were obtained immediately after artificial abortion. Soft villous material was cut away from connective tissue and vessels. Trypsin(0.25%)-Dnase I (0.15 U/ml) joint digestion and repeated purification were performed to generate a homogeneous population of trophoblast cells. IgG FcR on cultured human trophoblast cells were detected by flow cytometry. The well-developed cells during longitudinal phase were divided into 6 groups randomly, which were co-incubated respectively with HBsAg, HBsAg-anti-HBs complex, heat-inactivated HBsAg+ ?HBeAg+ , anti-HBc+ sera, cocultures of heat-inactivated HBsAg+N HBeAg+^ anti-HBc+ sera and sera with high titer of anti-HBs, heat-inactivated normal sera, and normal medium(blank control). Every 12h, the coverslips with cells confluent was harvested for anti-HBs immunohistochemical staining. Results Cells isolated as described in this paper comprised a highly purified villous trophoblast population and may be satisfactory for many experiments. Flow Cytometric detection results indicated that Fc Y R HI was highly expressed on the cultured trophoblast cells.The cells in normal human serum group and in blank control group werewell-developed.The cells in other groups were well-developed at 12h, 24h, displayed karyopyknosis and karyolysis at 48h, and exfoliated and died afterwards. After 12h and 24h co-incubation with different factors, HBsAg was detected to be positive in the cells co-incubated with HBsAg-anti-HBs complex, cocultures of heat-inactivated HBsAg+> HBeAg+>anti-HBc+ sera and sera with high titer of anti-HBs, and negtive in other cell groups.Conclusions Human trophoblast cells could intake HBsAg in the form of HBsAg-anti-HBs complex, instead of single HBsAg molecule. So, we postulated that, in the pregnant women infected with HBV, the HBsAg and anti-HBs in the blood formed HBsAg-anti-HBs complex, the Fc fragment of anti-HBs IgG bound with Fc Y R III on the trophoblast cells, through which endocytosis of HBV into trophoblast cells was mediated, so that HBV infection of trophoblast cells was induced and HBV intrauterine transmission occurred.