RNA干扰技术沉默TRAF6对脂多糖诱导肾小管上皮细胞表型转化及炎症因子表达的影响

摘要

Objective To study the expression of TNF receptor associated factor 6 (TRAF6) in human renal tubular epithelial cells induced by lipopolysaccharide silenced by RNA interference on cell phenotype transformation and expression of inflammatory cytokines.Methods TRAF6 small interference RNA (siRNA) by synthesis was transfected into HK-2 cells by cationic liposome method.HK-2 cells were divided into a blank control group,lipopolysaccharide group,negative control group and TRAF6 siRNA interference group.6,12 and 24 h after every group was cultured,the morphology of the cells was observed by inverted microscope and proliferation of cells was detected by Thiazolyl Blue Tetrazolium Bromide assay.The contents of interleukin-6 (IL-6) and tumour necrosis factor-a (TNF-α) in the cells were detected by enzyme linked immunosorbent assay and the expressions of a-smooth muscle actin (α-SMA) and TRAF6 rnRNA were detected by reverse transcription-PCR (RT-PCR).The expressions of TRAF6,α-SMA,p38,c-Jun N-terminal kinase-mitogen activated protein kinase (JNK MAPK) and nuclear factor-kappa-B (I-κB) inhibitor protein were detected by Western blot.Results TRAF6 siRNA significantly reduced TRAF6,α-SMA rnRNA and protein expression in HK-2 cells induced by lipopolysaccharide (P ＜ 0.05).By transfection,the proliferation of cells decreased,cytokines IL-6,TNF-α content decreased (P ＜ 0.05) and P38,JNK MAPK and I-κB protein phosphorylation levels were significantly reduced (P ＜ 0.05).Conclusion TRAF6 can mediate between LPS-induced tubular epithelial cell inflammatory response and cell phenotype transformation through MAPK-NF-κB signaling pathway.%目的 探讨RNA干扰技术沉默脂多糖诱导人肾小管上皮细胞HK-2中肿瘤坏死因子受体相关因子6(TRAF6)基因表达后,对HK-2细胞表型转化及炎症因子表达的影响.方法 应用阳离子脂质体法将设计合成的TRAF6小干扰RNA(siRNA)瞬时转染HK-2细胞.将HK-2细胞分为空白对照组、脂多糖组、阴性对照组和TRAF6 RNA干扰组.各组培养6、12、24h后,倒置显微镜观察细胞形态,噻唑蓝法检测细胞增殖,酶联免疫吸附测定法检测细胞中白细胞介素6(IL-6)、肿瘤坏死因子cα(TNF-α)的含量,实时荧光定量PCR法检测α-平滑肌肌动蛋白(α-SMA)、TRAF6 mRNA的含量,蛋白免疫印迹法检测TRAF6、α-SMA、p38、c-Jun氨基端激酶—丝裂原活化蛋白激酶(JNk MAPK)和核因子-κB抑制蛋白(NF-κB)的表达.结果 TRAF6 siRNA能显著降低脂多糖诱导HK-2细胞中TRAF6、α-SMA mRNA含量和蛋白的表达,TRAF6 siRNA转染后HK-2细胞的增殖能力减弱,细胞因子IL-6、TNF-α含量下降,p38、JNK MAPK和NF-κB蛋白的磷酸化水平均明显降低,均P＜0.05.结论 TRAF6可通过MAPK-NF-κB信号通路介导脂多糖诱导的肾小管上皮细胞炎症反应和细胞表型转化.