RNA干扰Ywhaz基因对高糖诱导的肾小球系膜细胞凋亡及炎症因子的影响  

Effect of RNAi with Ywhaz gene on apoptosis and inflammatory factors in glomerular mesangial cells induced by high glucose

作　　者：王颖怡[1] 李婧波[1] 张艳(指导)[1] 许德超[1,2] WANG Ying-Yi;LI Jing-Bo;ZHANG Yan;XU De-Chao(Nephrology Department,The Second Affiliated Hospital of Qiqihar Medical University,Qiqihar 161006,China)

机构地区：[1]齐齐哈尔医学院附属第二医院肾内科,齐齐哈尔161006 [2]齐齐哈尔医学院附属第二医院神经内五科,齐齐哈尔161006

出　　处：《中国免疫学杂志》2019年第13期1631-1635,共5页Chinese Journal of Immunology

摘　　要：目的:探究RNA干扰(RNAi)色氨酸5-单加氧酶激活蛋白(Ywhaz)基因对高糖诱导的肾小球系膜细胞增殖、凋亡的影响及可能作用机制。方法:在Lipofectamine 2000介导下,将siRNA阴性对照以及siRNA-Ywhaz分别转染高糖条件下培养的小鼠肾小球系膜细胞,以高糖未转染和正常培养的肾小球系膜细胞作为对照,实时定量PCR(RT-PCR)检测各组细胞中Ywhaz mRNA水平;噻唑蓝(MTT)实验检测细胞的增殖情况,流式细胞术检测细胞的凋亡率;RT-PCR检测各组细胞中炎症因子白细胞介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)的表达量;蛋白质印迹法(Western blot)检测Ywhaz蛋白、丝裂原活化蛋白激酶(MAPK)、磷酸化p38丝裂原活化蛋白激酶(p-p38 MAPK)和磷酸化丝裂原活化蛋白激酶-3(p-MKK3)的表达水平。结果: Ywhaz在高糖对照组细胞中的表达量显著高于正常对照组( P <0.05),RNA干扰后Ywhaz基因的表达量较高糖对照组显著降低( P <0.05);与高糖对照组相比,siRNA-Ywhaz组细胞的增殖能力明显降低( P <0.05),凋亡率显著增加( P <0.05),显著抑制IL-8和TNF-α以及p-p38 MAPK、p-MKK3的表达量( P <0.05)。结论:下调Ywhaz可降低p38 MAPK 信号通路关键蛋白 p-p38 MAPK、p-MKK3的表达以及炎症反应,从而抑制高糖诱导的肾小球系膜细胞增殖。Objective: To explore the effect of RNA interference(RNAi)tryptophan 5-monooxygenase-activating protein(Ywhaz)gene on proliferation and apoptosis of glomerular mesangial cells induced by high glucose and its possible mechanism. Methods: Mouse mesangial cells were cultured at high glucose concentration to mimics diabetes mellitus and transfected with siRNA negative controls and siRNA-Ywhaz of Lipofectamine 2000,using those untransfected and those transfected after culture at low glucose concentration as control.The expression of Ywhaz mRNA in cells of various groups were determined by real-time quantitative PCR(RT-PCR);the proliferation of cells was measured by MTT assay and the apoptotic rate was analyzed by flow cytometry;RT-PCR was used to detect the expression of inflammatory factors interleukin-8 (IL-8) and tumor necrosis factor-α(TNF-α) in each group,Western blot was used to detect the expression of Ywhaz protein,phosphorylated p38 mitogen-activated protein kinase (p-p38MAPK) and phosphorylated mitogen-activated protein kinase-3 (p-MKK3). Results: The expression of Ywhaz in the HG-control group was significantly higher than that in the normal control group( P <0.05),but the expression of Ywhaz gene in the siRNA-Ywhaz group was significantly lower( P <0.05).Compared with the HG-control group,siRNA-Ywhaz cells had the significantly effects on inhibiting proliferation( P <0.05),promoted cell apoptosis( P <0.05),decreased the levels of IL-8 and TNF-α,and inhibite the protein levels of p-p38 MAPK and p-MKK3( P <0.05). Conclusion: Down-regulation of Ywhaz can reduce the expression of p-p38 MAPK and p-MKK3 in p38 MAPK signaling pathway and inflammatory response,thus inhibite the proliferation of glomerular mesangial cells induced by high glucose.

关 键 词：肾小球系膜细胞 糖尿病肾病 增殖 Ywhaz 

分 类 号：R285.5[医药卫生—中药学]