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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。
Objective To investigate whether hepatitis B immunoglobulin (HBIG) could block the infection of hepatitis B virus (HBV) on trophoblasts in vitro under in vitro conditions. Methods Placental trophoblast cells were transferred to 6-well plates and cultured in DMEM medium containing 10% fetal bovine serum at 37 ℃ in 5% CO2 incubator. After 24 hours, the cells were blocked with HBV infection. Group B: 2% DMEM3ml + HBV-positive serum 0.5ml (HBV positive sera were incubated with 80U HBIG for 30min at 37 ℃) . Group C: 2% DMEM3ml + HBV-positive serum 0.5ml (HBV positive sera were incubated with 40U HBIG for 30min at 37 ° C); Group D: 2% DMEM3ml and 40u HBIG were incubated at 37 ° C After 30min + HBV positive serum 0.5ml; E group: 2% DMEM3ml + 40U HBIG; F group: 2% DMEM3ml + HBV negative serum 0.5ml. After 24 hours, the medium in each well was removed, and each cell culture well was thoroughly washed with 0.01 M phosphate-buffered saline (PBS). 2% DMEM 4 ml was further cultured and collected every 12 h (12 to 84 h) Cell culture supernatants in culture wells were detected by enzyme-linked immunosorbent assay HBsAg levels in cell culture supernatant [expressed as absorbance (A)], PCR detection of cell culture supernatant HBVDNA. Results The levels of HBsAg in cell culture supernatants of groups A, B, C, D, E and F before PBS washing were respectively 2.697,0.040,0.102,0.098,0.036,0.040 ; HBVDNA was positive in cell culture supernatant of groups A, B, C and D; HBVDNA was negative in groups E and F; The mean values of HBsAg in supernatant of group A, B, C, D, E and F cultured for 12-84 h were 1 · 550 ± 0 · 270,0 · 032 ± 0 · 016,0 · 100 ± 0 · 0 · 087,0 · 052 ± 0 · 044,0 · 034 ± 0 · 020,0 · 034 ± 0 · 022; The level of HBsAg in group A was significantly different from that in group B, C, D, E and F (P <0 · 01). 84h cell culture supernatant, A group HBVDNA positive; B, C, D, E, F group HBVDNA negative. Conclusion In vitro studies have shown that HBIG can effectively block the infection of placental trophoblast cells.