经HBx转染后的肾小管上皮细胞对T细胞激活、分化的影响

Effect of renal tubular epithelial cells with hepatitis B virus x gene on mediating T-cell activationand differentiation

目的探讨乙型肝炎病毒x基因（hepatitis B virusx，HBx）转染人近端肾小管上皮细胞系（HK－2）后其对T细胞激活、分化的影响。方法分4组：实验组（即转染HBx质粒的HK－2细胞＋CD4＋T细胞）、阴性对照组（即转染HBx空载质粒的HK－2细胞＋CD4＋T细胞）、单独培养组（即CD4＋T细胞）和空白对照组（未处理HK－2细胞＋CD4＋T细胞）。体外培养HK－2，用分子克隆的方法构建pcDNA3．1－myc—HBx质粒，采用脂质体转染法瞬时转染HK．2细胞，实时荧光定量PCR及Western印迹法验证HBx在HK－2细胞中的表达。免疫磁珠法分选健康志愿者外周血CD4＋T细胞，分别与HK－2细胞共培养，流式细胞术检测HK－2细胞共刺激分子CD40的表达、CD4＋T细胞CD40配体（CD40L）表达及细胞周期情况，ELISA检测各组细胞培养上清中Thl型细胞因子IFN-r及Th2型细胞因子IL-4水平。结果HK-2细胞经转染pcDNA3．1-myc—HBx质粒后，可高表达HBx。转染HBx基因后HK-2细胞CD40表达显著上调（P〈0．01）；与对照组相比，实验组CD4’T细胞表面CD40L表达也显著增加（P〈0．01），S期与G2／M期细胞数之和增多（P〈0．01），培养上清中IFN-1水平升高（P〈0．05），IL-4水平降低（P〈0．05）。结论肾小管上皮细胞经HBx转染后，其共刺激分子表达上调，并促进CD4＋T细胞增殖，诱导CD4＋T细胞向Thl方向分化。由此推测，乙肝相关性肾炎肾组织免疫损伤所导致病变的持续进展也可能与此因素的参与相关。

Objective To investigate the effect of renal tubular epithelial cells with hepatitis B virus x （HBx） gene on mediating T-cell activation and differentiation. Methods The eukaryotic vector peDNA3.1- myc- HBx containing HBx gene was transiently transfected into HK- 2 cells by lipofectamine mediation. The expression of HBx was confirmed by real-time PCR and Western blotting. CD4＋ T cell in peripheral blood of health volunteer was isolated and purified by immunomagnetic beads. Subsequently, purified CD4＋ T cells were co-cultured with HK-2 cells. Flow cytometry was used to detecte the expression of CD40 of HK- 2 and the expression of CD40L and cell cycle of CD4＋ T cells. ELISA assay was used to quantify the IFN-r and IL- 4 in co- culture supernatant. The experiment was divided into four groups： experimental group（HK- 2 cells with HBx ＋ CD4 ＋T cells）, negative control group （HK- 2 cells with pcDNA3.1-myc ＋CD4＋T cells）, blank control group （HK-2 cells ＋CD4＋T cells） and separate culture group （CD4＋T cells）. Results Real-time PCR and Western blotting results verified that HBx was successfullyexpressed in HK- 2 cells after transfection. After transfection of HBx gene, the expression of CD40 was significantly increased on HK- 2 cells（P 〈 0.01）. Compared with control groups, the expression level of CD40L and the percentage of cells at S and G2/M phase increased（all P 〈 0.01）. Meanwhile, the level of IFN-r in the supernatant was higher（P〈 0.05）, but the level of IL-4 was lower in experimental group（P 〈 0.05）. Conclusions Over-expression of HBx gene may up-regulate the expression of costimulatory molecules CD40 of renal tubular epithelial cells, which may active CD4＋ T cells, promote the proliferation of CD4＋ T cells and up-regulate Thl-type cytokines. These events may induce immune injury of renal in hepatitis B virus-associated glomerulonephritis.