TNFα对HBV体外感染人绒毛膜癌JEG Ⅲ细胞的促进作用

Enhancement effect of TNFα on hepatitis B virus in vitro infection of human choriocarcinoma JEGIII cells

目的:检测TNF-α存在条件下HBV体外感染人绒毛膜癌细胞的情况,为HBV宫内传播机制的研究提供细胞学基础. 方法:在TNF-α存在条件下,绒毛膜癌JEGIII细胞与HBV阳性血清(2×1012HBV DNA/L)共同孵育.感染后24 h,胰酶常规消化细胞,PBS充分洗涤,直至最后一遍洗液ELISA检测HBsAg阴性,重新接种细胞,加新鲜培养液继续培养,每隔12 h,收集培养标本,分别用Western blotting,免疫细胞化学方法,透射电镜检测培养物中HBV标志物. 结果:HBV阳性血清感染JEGIII细胞36 h后,各时间点收集的细胞上清标本中Western blotting均检测到阳性的HBsAg条带,免疫细胞化学方法检测细胞铺片发现,在TNF-α存在的感染环境下,HBsAg呈阳性或强阳性表达,与TNF-α不存在的感染环境下相比有显著性差别(20.40±4016 vs 7.40±1.82,P<0.01),而且HbsAg主要位于胞膜和/或胞质,透射电镜下,细胞扩张的粗面内质网腔内发现有杆状HBsAg颗粒. 结论:HBV体外可以成功感染人绒毛膜癌细胞.本体外细胞感染模型是深入研究HBV宫内传播机制的关键.

AIM: To study the in vitro infection of human choriocarci-noma JEGIII cells by hepatitis B virus (HBV) in the presence of TNF-α, and to provide some clues for the mechanism responsible for HBV intrauterine transmission. METHODS: Human choriocarcinoma-derived JEGⅢ cells were exposed to HBV (2 × 1012 HBV DNA/L) in the presence of TNF-α. After an overnight incubation, the cells were then trypsinized, extensively washed with PBS till the last washings were negative for HbsAg as identified by ELISA. The cells were re-inoculated and kept on subculturing with fresh medium. Then the specimens were collected at an interval of 12 hours. HBsAg in supernatants and cells was detected by Western blotting, immunohistochemistry and transmission electron microscopy. RESULTS: After JEGIII cells were infected with HBV for 36 hours, HBsAg was positive in supernatants collected at different time points. The level of HBsAg was significantly higher in JEGIII cells infected by HBV in the presence of TNF-α than that in the absence of TNF-α (20.40±4016 vs 7.40±1.82, P<0.01). HBsAg was mainly located in the cytolemma and/or the cytoplasm. Under electron microscope, HBsAg particles were observed in the dilated cisterns of rough surfaced endoplasmic reticulum of the cells. CONCLUSION: Human choriocarcinoma JEGIII cells can be susceptibly infected by HBV in the presence of TNF-α in vitro, and this may provide some clues for further studying HBV intrauterine transmission.