乙型肝炎病毒感染肾小管上皮细胞Toll样受体4的表达及其作用

The expression and role of Toll receptor 4 in renal tubular epithelial cells in hepatitis B virus infection

目的 探讨HBV感染人近端肾小管上皮细胞系HK-2后对其表达Toll样受体4（TLR4）的影响,并观察TLR4抗HBV感染的生物学作用.方法 收集HBV DNA拷贝在107-102/ml的患者血清,通过显微镜及免疫荧光法观察HBV阳性血清感染HK-2前、后细胞形态及α抗平滑肌抗体（α-SMA）的变化,应用MTT法检测不同浓度TLR4刺激因子（LPS）及TLR4抑制因子（CLI-095）对HK-2细胞增殖的影响.选取10 μL/ml脂多糖（LPS）及5μg/ml CLI-095作用于HBV感染的HK-2细胞,通过细胞免疫荧光技术及免疫印迹法检测HK-2细胞内TLR4蛋白的变化,ELISA法和荧光定量PCR法观测各组细胞上清液中HBsAg、HBeAg和HBV DNA含量的变化.结果 HBV分别感染HK-2细胞12 h和24 h后,随感染时间延长,细胞形态变得不规则,数量也减少.α-SMA的表达水平与感染24 h后相比,在HBV感染12 h后表达最多.LPS浓度在小于10μg/ml范围内,HBV感染HK-2细胞24 h后其增殖程度与剂量呈正相关,与CLI-095浓度呈负相关（P＜0.05）.LPS组HK-2细胞TLR4蛋白的表达高于CLI-095组,其上清液中HBV DNA水平及HBsAg、HBeAg表达水平较CLI-095组降低.结论 TLR4可能通过免疫炎症反应参与抑制HK-2细胞中的HBV复制,当HBV感染肾组织细胞时可发挥抗病毒作用.

Objective To explore the expression and role of Toll receptor 4（TLR4）in human proximal tubular epithelial cell line HK-2,infected by HBV. Methods The serum of HBV DNA copies between 107-108/ml was collected. Before and after infected by HBV DNA positive serum. the HK-2 cells＇ morphology and the expression of α-smooth muscle actin（α-SMA）were observed by microscopy and immunofluorescence, and the effects of different concentrations of lipopolysaccharides（U）S.TLR4-stimulating factor）and CLI-095（TLR4 Inhibitor）on the proliferation rate of HK-2 cells were observed by MTT assays. After HBV serum and 10μg/ml LPS and 5μl/ml CLI-095 acted on HK-2 cells,TLR4 protein expression was measured by immunofluorescence and Western-blotting assay, and HBsAg and HBeAg in cell culture medium were detected by ELISA. and HBV DNA copies by fluorescence quantitative PCR. Results The longer HBV infected HK-2 cells, the more irregular of the cells＇ shape, the fewer number of the cells were left. But compared with HBV infected after 24 hours, α-SMA was more expressed after HBV infected 12 hours. After infected by HBV serum in 24 hours.HK-2 cells＇ proliferation rate was positively correlation in a dose range of LPS, but was negatively correlated with the CLI-095（P＜0.05＝.The levels of HBsAg and HBeAg in cell culture medium were largest when the LPS concentration was at 10μg/ml and CLI-095 at 5μg/ml.The expression of TLR4 significantly increased in HK-2 cells treated with LPS compared with those with CLI-095.but HBV DNA levels and HBsAg and HBeAg expression levels were lower. Conclusions HBV infection may promote cell transdifferentiation and cell injury. The stimulation of HK-2 infected with HBV by LPS may upregulate the expression of TLR4 and reduce the copies of HBV DNA.