乙型肝炎病毒对肾小管上皮细胞直接作用的实验研究

目的：探讨乙型肝炎病毒（HBV）在体外对人肾小管上皮细胞（HKC）的直接致病作用。方法：①HBV体外感染HKC：培养的HKC传至6孔板中，接种密度为1×10^6个／ml，孵育24h后分受感染和阴性对照两组。受感染组加入0．5ml HepG2．2．15细胞上清液（含有HBV）；阴性对照组加入0．5ml 10％FCS—DMEM／F—12培养液。两组HKC继续孵育72h，消化，离心沉淀，PBS清洗，实时荧光定量聚合酶链反应（FQ—PCR）检测两组HKC中的HBV DNA含量。②HBV对体外培养的HKC细胞直接作用：HKC细胞传至24孔板中，接种密度为1×10^5个／ml，受感染组分三个亚组，分别加入100μl、200μl和300μl的感染用HepG2．2．15细胞上清液；阴性对照组为接种HKC后加入培养液常规培养；阳性对照组为HepG2．2．15细胞上清液。各组的每孔终体积均为2ml，每组设3复孔。孵育72h后收集各孔上清液，ELISA法检测转化生长因子β1（TGF-β1）的表达。结果：①受感染组HKC的基因组中检测到HBV DNA。②受感染组的三个亚组细胞培养上清的TGF-β1与阴性对照组比较差异均有显著性。结论：HBV可感染HKC并促进其分泌TGF-β1。

Experimental study of direct pathogenic effects of hepatitis B virus on human tubular kidney cells

Objective： To investigate the direct pathogenic effects of Hepatitis B virus （HBV） on human tubular kidney cells （HKCs） in vitro. Methods： HKCs were infected by HBV： HKCs were seeded into six well cluster dishes, at 1×10^6 cells per ml. 24 hours later, HKCs were divided into control group and infected group. Cells of infected group were cultured with 0.5 ml supernatant of HepG2.2.15 cells, while 0.5 ml 10% FCS-DMEM/F-12 was used in control group. 72 h later, all of the HKCs were removed and then washed with PBS. HBV DNA was detected by fluorescent quantitation polymerase chain reaction （FQ-PCR）. Direct pathogenic effects of HBV on HKCs： HKCs were seeded into twenty four well cluster dishes, at 1×10^5 cells per ml. Negative control group, positive control group and infected group were established. HKCs of infected subgroups were cultured with 100 μl. 200 μl and 300 μl supernatant of HepG2.2.15 cells, respectively. Cells of negative control group were cultured with FCS-DMEM/F-12. Cells of positive control group were cultured with supernatant of HepG2.2.15. Supernatant from each well was collected after 72 hours. The expression of TGF-β1 was detected with ELISA, Results： HBV DNA was observed in the genome of HKC. In the HKC, persistent infection of HBV enhanced the expression of transforming growth factor β1 （TGF-β1 ）. Conclusion： Human tubular epithelial cell is susceptible to HBV and HBV infection can enhance the expression of TGF-β1.