过表达Sirt1对高糖诱导的人肾小球系膜细胞增殖与TGF-β1表达的影响  

Effects of over-expressed Sirt1 on high glucose-induced proliferation and TGF- β1 expression in human glomerular mesangial cells

作　　者：王星云[1] 朱剑[1] 蒋兰兰[1] 丁波[1] 马建华[1] 

机构地区：[1]南京医科大学附属南京医院内分泌科,江苏南京210012

出　　处：《南京医科大学学报（自然科学版）》2015年第4期480-484,509,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金(81100576);南京市卫生局杰出青年基金(JQX12006);南京市卫生青年人才培养工程基金(宁卫科[2011]42号)

摘　　要：目的 :探讨过表达沉默信息调节因子1(silent information regulator 1,Sirt1)对高糖诱导的人肾小球系膜细胞增殖与转化生长因子-β1(TGF-β1)表达的影响。方法:体外培养人肾小球系膜细胞(HMC),利用Sirt1重组慢病毒载体感染细胞获得过表达Sirt1蛋白的人肾小球系膜细胞并分为4组:1NG+LV-CTL组:正常葡萄糖(5.5 mmol/L)+空白对照慢病毒感染;2HG+LV-CTL组:高糖(30 mmol/L)+空白对照慢病毒感染;3NG+LV-Sirt1组:正常葡萄糖+Sirt1慢病毒感染;4HG+LV-Sirt1组:高糖+Sirt1慢病毒感染;再设立3组未感染细胞作为对照,分别为:1NG组(正常葡萄糖);2NM组(正常糖+甘露醇组24.5 mmol/L);3 HG组(高糖处理),培养不同时间后以CCK-8法检测细胞增殖情况;实时定PCR和Western blot法检测各组细胞TGF-β1m RNA和蛋白表达水平。结果:1通过重组慢病毒感染,嘌呤霉素筛选,获取稳定过表达Sirt1蛋白的人肾小球系膜细胞系;2与NG组相比较,各时间点HG、HG+LV-CTL、HG+LV-Sirt1组CCK8的吸光值均明显升高(P均<0.01)。处理24、48 h后,HG+LVSirt1组的吸光值明显低于HG、HG+LV-CTL组。3与NG组相比较,HG、HG+LV-CTL、HG+LV-Sirt1组TGF-β1 m RNA以及蛋白水平明显上调,差异均有显著性(P均<0.01);HG+LV-Sirt1组的TGF-β1 m RNA以及蛋白水平明显低于HG、HG+LV-CTL组(P均<0.01);而NG、NM、NG+LV-CTL及NG+LV Sirt1组间相比较,无统计学差异(P>0.05)。结论 :过表达Sirt1能够抑制高糖诱导的人肾小球系膜细胞增殖与TGF-β1表达,Sirt1可能成为防治糖尿病肾病的作用靶点。Objective：To investigate the effects of silent information regulator 1 （Sirtl） over-expression on high glucose-induced proliferation and transforming growth factor-β1 （TGF-β1 ） expression in human glomernlar mesangial cells （HMCs）. Methods： HMCs were cultured in vitro. The HMCs were transfected with either lentiviral vectors containing the Sirtl cDNA（LV-Sirtl） or empty vectors （LV-CTL）. The expression of Sirtl protein was detected by Western blot. Those HMCs transfected with lentiviral vectors were divided into 4 groups including normal glucose（5.5 mmol/L） plus LV-CTL（NG＋LV-CTL）, high glucose （30 mmol/L） plus LV-CTEHG＋ LV- CTL）, normal glucose plus LV- Sirtl （NG＋LV-Sirtl）, and high glucose plus LV-Sirtl （HG＋LV-Sirtl） groups. Those untransfected HMCs were cultured at normal glucose （NG）, normal glucose plus mannitol （5.5 mmol/L glucose ＋ 24.5 mmol/L mannitol ） （NM） or high glucose （30mmol/L） （HG） as control. After treatment for different periods, the cell proliferation of each group was examined using CCK-8 detection kit, and the levels of mRNA and protein of TGF-β1 from each group were measured through real-time polymerase chain reaction （PCR） and Western blot, respectively. Results： The HMCs, which was stably over-expressed Sirtl protein, was obtained after recombinant lentivirus infection and puromycin selection. Compared with the NG group, the proliferation levels of HMCs from the HG, HG＋LV-CTL and HG＋LV-Sirtl groups on 12, 24 and 48 hours were increased significantly （each P 〈 0.01）. However, the proliferation level of the HG＋LV-Sirtl group is significantly lower than that in the HG and HG＋ LV-CTL groups. As compared to counterparts of the HG and HG＋ LV-CTL groups, the levels of mRNA and protein of TGF-β1 from the HG＋LV Sirtl group were decreased significantly （each P 〈 0.01）. The levels of mRNA and protein of TGF-β1 from the HG treated groups were higher than those of the NG group（each P 〈 0.01

关 键 词：沉默信息调节因子1 高糖 人肾小球系膜细胞 转化生长因子-β1 

分 类 号：R587.2[医药卫生—内分泌]