乙型肝炎病毒阳性血清体外感染Hep G2细胞的实验研究

目的　建立HBV阳性血清体外感染HepG2 细胞的实验方法。方法　培养HepG2 细胞传至6孔板中,2 4h后进行HBV阳性血清体外感染HepG2 细胞的实验。感染组用HBV阳性血清,阴性对照组用HBV阴性血清,空白对照组用DMEM培养基。实验开始后HepG2 细胞继续孵育2 4h ,而后用0 0 1mol LPBS清洗8次后加入2 %DMEM培养液。收集PBS第8次洗液,收集PBS洗后每隔12h各孔细胞培养上清。ELISA检测细胞培养上清中的HBsAg。PCR检测细胞培养上清和HepG2 细胞中的HBVDNA。结果　感染组在PBS洗后12h的细胞培养上清中ELISA检测HBsAg呈阳性。PCR检测显示感染组细胞培养上清和HepG2 细胞中HBVDNA呈阳性,阴性对照组和空白对照组HBVDNA呈阴性。结论　HBV阳性血清进行HBV感染体外培养HepG2 细胞是可行的。

In vitro infection of human hepatoma (Hep G2) cell line by hepatitis B virus positive serum

OBJECTIVE: To establish a culture system of HBV positive serum infected Hep G2 cells in vitro. METHODS: Hep G2 cells were seeded into six-well cluster dishes, at 1 x 10(-6) cells per well and incubated with 3 ml 10% fetal calf serum/ Dubecco's modified Eagle's medium (10% FCS/DMEM) at 37 degrees in 5% CO2 air. At 24 h after plating, infection group Hep G2 cells were cultured with 0.5 ml HBV positive serum, in control group HBV negative serum was used, 24 h later the inoculums was removed. The cells were then extensively washed with 0.01 mol/L phosphate-buffered saline (PBS). After washing with PBS, 4 ml 2% FCS/DMEM were added to each well and the medium was collected every 12 h. ELISA method was used to detect HBsAg in culture medium. HBV DNA in cells and culture medium was detected by PCR. RESULTS: In infection group, HBsAg could be detected from cell culture medium from 12 h (after PBS washed) to 84 h. HBV DNA could be detected by PCR in culture medium and cells. CONCLUSION: Infection of Hep G2 cells by HBV positive serum is feasible.