乙型肝炎免疫球蛋白阻断胎盘滋养细胞感染乙型肝炎病毒的实验研究

目的探讨在体外条件下,乙型肝炎免疫球蛋白(HBIG)能否阻断乙型肝炎病毒(HBV)对胎盘滋养细胞的感染。方法将胎盘滋养细胞传至6孔板中,用含10%新生牛血清的DMEM培养基在37℃、5%CO2孵箱中培养。细胞传入24h后进行HBV感染阻断实验。实验分为A组:2%DMEM3ml+HBV阳性血清0·5ml;B组:2%DMEM3ml+HBV阳性血清0·5ml(HBV阳性血清加入前,先与80U的HBIG在37℃条件下孵育30min)。C组:2%DMEM3ml+HBV阳性血清0·5ml(HBV阳性血清加入前,先与40U的HBIG在37℃条件下孵育30min);D组:2%DMEM3ml与40U的HBIG在37℃条件下孵育30min后+HBV阳性血清0·5ml;E组:2%DMEM3ml+40U的HBIG;F组:2%DMEM3ml+HBV阴性血清0·5ml。24h后,移去各孔中的培养液后,用0·01mol/L的磷酸盐缓冲液(PBS)彻底清洗各细胞培养孔,2%DMEM4ml继续培养,每隔12h(12~84h)收集各细胞培养孔中的培养上清液,应用酶联免疫吸附实验检测细胞培养上清液中的HBsAg水平[以吸光度(A)值表示],PCR检测细胞培养上清液中HBVDNA。结果PBS清洗前A、B、C、D、E、F组细胞培养上清液中的HBsAg水平分别为2·697、0·040、0·102、0·198、0·036、0·040;A、B、C、D组细胞培养上清液中HBVDNA为阳性,E、F组HBVDNA为阴性。A、B、C、D、E、F组培养12~84h上清液中HBsAg水平均值分别为1·550±0·270、0·032±0·016、0·100±0·087、0·052±0·044、0·034±0·020、0·034±0·022;A组HBsAg水平与B、C、D、E、F组比较,差异有统计学意义(P<0·01)。84h的细胞培养上清液,A组HBVDNA阳性;B、C、D、E、F组HBVDNA阴性。结论体外实验研究表明,HBIG可以有效阻断HBV对胎盘滋养细胞的感染。

Hepatitis B immunoglobulins blocking hepatitis B virus infection of trophoblast cell culture in vitro

OBJECTIVE: To determine the role of hepatitis B Immunoglobulins (HBIG) in blocking hepatitis B virus (HBV) infection of trophoblast cell culture in vitro. METHODS: Trophoblast cells were placed in the six-well cluster dishes and incubated with 10% fetal calf serum/Dubecco's modified Eagle's Medium (10% FCS DMEM) at 37 degrees C with 5% CO2 in air. At 24 h after plating cells were subjected to experiment. Group A: cells were cultured with 0.5 ml HBV positive serum plus 3 ml 2% FCS DMEM; Group B: cells were cultured with 3 ml 2% FCS DMEM plus 0.5 ml HBV positive serum pretreated with 80 U HBIG for 30 min at 37 degrees C; Group C: cells were cultured with 3 ml 2% FCS DMEM plus 0.5 ml HBV positive serum pretreated with 40 U HBIG for 30 min at 37 degrees C; Group D: cells were cultured with 3 ml 2% FCS DMEM plus 40 U HBIG for 30 min before 0.5 ml HBV positive serum was added; Group E: cells were cultured with 40 U HBIG plus 3 ml 2% FCS DMEM; Group F: cells were cultured with HBV negative serum plus 3 ml 2% FCS DMEM. Twenty-four hours later the inoculums were removed, and the cells were extensively washed with 0.01 mol/L phosphate-buffered saline (PBS). After PBS washing, 4 ml 2% FCS DMEM was added to each well and the medium was collected every 12 hours. Enzyme-Linked Immunosorbent Assay (ELISA) method was used to detect HBsAg in culture medium (absorption value, A). HBV DNA in cell culture medium was detected by polymerase chain reaction (PCR). RESULTS: Before PBS washing, the A value of groups A, B, C, D, E, F were 2.697, 0.040, 0.102, 0.198, 0.036, 0.040 respectively. The cell culture medium in groups of A, B, C, and D were HBV DNA positive, groups of E, F were HBV DNA negative. From 12 hours to 84 hours, the average A value of groups A, B, C, D, E and F was 1.55 +/- 0.27, 0.032 +/- 0.016, 0.100 +/- 0.087, 0.052 +/- 0.044, 0.034 +/- 0.020, 0.034 +/- 0.022 respectively. The A value of groups A was significantly higher than those of other groups (P < 0.01). Cell culture medium at 84 hours of group A was HBV DNA positive and those of group B, C, D, E, F were HBV DNA negative. CONCLUSION: HBIG could effectively block HBV infection of trophoblast cell culture in vitro.