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目的:探讨乙型肝炎病毒x基因(HBx)转染人近端肾小管上皮细胞系(HK-2细胞)后对其凋亡及细胞因子分泌的影响及可能机制。方法:体外培养HK-2细胞,用分子克隆法构建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(RT-PCR)及蛋白印迹法验证HBx在HK-2细胞中的表达。以未转染质粒组和转染空载质粒pcDNA3.1/myc组作为对照,显微镜观察转染HBx基因后HK-2细胞形态,RT-PCR检测各组细胞Toll样受体4(TLR4)mRNA水平,蛋白印迹法检测各组细胞TLR4的表达,流式细胞术检测各组细胞凋亡情况,ELISA检测细胞培养上清中白细胞介素4(IL-4)、干扰素γ(IFN-γ)水平。结果:转染pcDNA3.1/myc-HBx质粒后的HK-2细胞中HBxmRNA和蛋白表达水平明显增加,证实转染成功;转染HBx基因组细胞形态不规则,细胞状态受损;与对照组相比,转染HBx基因组TLR4的表达水平明显上调(P<0.05),凋亡细胞数量明显增多(P<0.05),细胞上清液中IFN-γ水平增加,但IL-4水平降低。结论:经构建pcDNA/myc-HBx质粒并转染肾小管上皮细胞可成功制备乙肝病毒细胞感染模型,由此导致的肾小管上皮细胞凋亡可能与HBx引起肾小管上皮细胞TLR4的表达上调有关,HBx亦能导致细胞因子分泌紊乱。
Objective: To investigate the effects of hepatitis B virus x gene (HBx) on the apoptosis and cytokine secretion of human proximal tubular epithelial cell line (HK-2) and its possible mechanism. Methods: HK-2 cells were cultured in vitro. The pcDNA3.1 / myc-HBX plasmids were constructed by molecular cloning. The transfected HK-2 cells were transfected with HK-2 cells by lipofectamine. Real-time fluorescence quantitative PCR (RT- Method to verify the expression of HBx in HK-2 cells. The untransfected plasmid group and pcDNA3.1 / myc transfected plasmid were used as a control. The morphological changes of HK-2 cells were observed by microscope after transfection with HBx gene. Toll-like receptor 4 (TLR4) mRNA The expression of TLR4 was detected by Western blotting and flow cytometry. The levels of interleukin-4 (IL-4), interferon-γ (IFN-γ) Level. Results: The expression of HBx mRNA and protein in HK-2 cells transfected with pcDNA3.1 / myc-HBx plasmid was significantly increased, which confirmed that the transfection was successful. The morphological changes of HBx-infected cells were impaired, (P <0.05). The number of apoptotic cells was significantly increased (P <0.05), and the level of IFN-γ in the cell supernatant was increased, but the level of IL-4 was decreased. CONCLUSION: Hepatitis B cell infection model can be successfully prepared by constructing pcDNA / myc-HBx plasmid and transfecting into renal tubular epithelial cells. The apoptosis of renal tubular epithelial cells may be related to the up-regulation of TLR4 induced by HBx in renal tubular epithelial cells. HBx can also cause cytokine secretion disorders.