Sirt1对高糖诱导的人肾小球系膜细胞增殖与TGF-β1表达的影响及其机制

发布时间：2018-05-13 17:15

本文选题：肾小球系膜细胞 + 糖尿病肾病　；参考：《南京医科大学》2015年硕士论文

【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(Human glomerular mesangial cell,HMC)增殖与转化生长因子-β1(TGF-β1)表达的影响,并进一步探究Sirt1对高糖诱导效应影响的可能机制。方法(1)体外培养人肾小球系膜细胞(HMCs),利用Sirt1、Sirt1干扰(Sirt1-RNAi)或相应对照重组慢病毒载体感染细胞获得过表达Sirt1蛋白或knock down Sirt1的人肾小球系膜细胞;(2)慢病毒感染的HMCs分为8组:(1)正糖+空载慢病毒感染组(NG+LV-CTL),(2)高糖+空载慢病毒感染组(HG+LV-CTL),(3)正糖+Sirt1慢病毒感染组(NG+LV-Sirt1)(4)高糖+Sirt1慢病毒感染组(HG+LV-Sirt1)(5)正糖+无义RNAi病毒感染组(NG+LV-CTL-RNAi),(6)高糖+无义RNAi病毒感染组(HG+LV-CTL-RNAi)(7)正糖+Sirt1-RNAi病毒感染组(NG+LV-Sirt1-RNAi)(8)高糖+Sirt1-RNAi病毒感染组(HG+LV-Sirt1-RNAi);再设立3组未感染细胞作为对照,分别为(1)正常对照组(NG,葡萄糖5.5 mmol/L)、(2)正糖+甘露醇组(NM,5.5mmol/L葡萄糖+24.5mmol/L甘露醇),(3)高糖处理组(HG,葡萄糖浓度30 mmol/L),培养不同时间后以CCK-8法检测细胞增殖情况;实时定量PCR法检测TGF-β1的mRNA水平,Western blot法检测各处理组细胞TGF-β1蛋白表达水平;(3)低糖、高糖处理HMC细胞不同时间后,通过Western blot或免疫共沉淀+Western blot检测STAT1、STAT3磷酸化、乙酰化水平;(4)通过Western blot或免疫共沉淀+Western blot检测正糖、高糖处理24小时后的LV-CTL、LV-Sirt1、LV-Sirt1-RNAi组STAT1磷酸化、乙酰化水平;(5)免疫共沉淀检测内源性Sirt1与STAT1相互结合作用。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和Sirt1干扰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、HG+LV CTL-RNAi、HG+LV-Sirt1-RNAi组CCK8的OD检测值均明显升高(P均0.05)。HG处理24h、48h后,HG+LV-Sirt1组的OD值明显低于HG、HG+LV-CTL组,HG+LV-Sirt1-RNAi组的OD值明显高于HG、HG+LV-CTL组;与NG组相比较,HG、HG+LV-CTL、HG+LV-Sirt1组TGF-β1mRNA以及蛋白水平明显上调,差异均有显著性(P均0.05);HG+LV-Sirt1组TGF-β1 mRNA以及蛋白水平明显低于HG、HG+LV-CTL组,而LV-Sirt1-RNAi组的TGF-β1 mRNA以及蛋白水平明显高于HG、HG+LV-CTL-RNAi组(P均0.05);NG、NM、NG+LV-CTL及NG+LV-Sirt1、NG+LV-CTL-RNAi组间相比较,无统计学差异;(3)高糖以时间依赖性的方式增加STAT1及STAT3磷酸化、乙酰化水平;(4)与NG+LV-CTL组相比,HG+LV-CTL组、HG+LV-Sirt1-RNAi组的STAT1磷酸化、乙酰化水平均升高,与HG+LV-CTL组相比较,HG+LV-Sirt1组STAT1乙酰化水平下降,HG+LV-Sirt1-RNAi组STAT1乙酰化水平升高(P均0.05),HG+LV-CTL组、HG+LV-Sirt1组和HG+LV-Sirt1-RNAi组的磷酸化水平无明显差异(P0.05);(5)正向、反向免疫共沉淀均能够检测到Sirt1蛋白与STAT1蛋白间的结合。结论过表达Sirt1水平能够抑制高糖诱导的人肾小球系膜细胞增殖、TGF-β1表达以及STAT1、STAT3的乙酰化水平,相反,沉默Sirt1的表达增强了高糖的处理效应;Sirt1可能是通过去乙酰化STAT1而发挥其拮抗高糖诱导效应的作用;Sirt1可能成为防治糖尿病肾病的作用靶点。
[Abstract]:Objective to investigate the effect of silent information regulator 1 (Sirt1) on the proliferation of human glomerular mesangial cells (Human glomerular mesangial cell, HMC) and the expression of transforming growth factor - beta 1 (TGF- beta 1) induced by high glucose, and to further explore the possible mechanism of Sirt1 on the effect of high glucose induction. Method (1) Cultured human glomerular mesangial cells (HMCs), using Sirt1, Sirt1 interference (Sirt1-RNAi) or corresponding recombinant lentivirus vector infected cells to obtain human glomerular mesangial cells expressing Sirt1 protein or knock down Sirt1; (2) the HMCs of lentivirus infection is divided into 8 groups: (1) positive sugar + nodTo lentivirus infection group (NG+LV-CTL), (2) high sugar + empty load. Virus infection group (HG+LV-CTL), (3) positive sugar +Sirt1 lentivirus infection group (NG+LV-Sirt1) (4) high sugar +Sirt1 lentivirus infection group (HG+LV-Sirt1) (5) positive sugar + non sense RNAi virus infection group (NG+LV-CTL-RNAi), (6) high sugar + non sense RNAi virus infection group (HG+ LV-CTL-RNAi) (7) positive sugar +Sirt1-RNAi virus infection group (NG+LV-Sirt1-RNAi) (8) high sugar sickness disease The virus infection group (HG+LV-Sirt1-RNAi); 3 groups of uninfected cells were set up as control, respectively (1) normal control group (NG, glucose 5.5 mmol/L), (2) positive sugar + mannitol group (NM, 5.5mmol/L glucose +24.5mmol/L mannitol), (3) high sugar treatment group (HG, glucose 30 mmol/L), and the cell proliferation was detected by CCK-8 method after different time; The mRNA level of TGF- beta 1 was detected by quantitative PCR, and TGF- beta 1 protein expression level was detected by Western blot method. (3) low sugar and high sugar treated HMC cells at different time, STAT1, phosphorylation and acetylation level were detected by Western blot or immunoprecipitation +Western blot; (4) LV-CTL, LV-Sirt1, LV-Sirt1-RNAi group, STAT1 phosphorylation and acetylation level after 24 hours of high glucose treatment; (5) immunoprecipitation was used to detect the interaction of endogenous Sirt1 and STAT1. Results (1) human glomerular mesangial cell lines were obtained by recombinant lentivirus infection, purinomycin screening, and the interference of Sirt1 and Sirt1 were obtained; (2) Compared with the NG group, the OD detection value of each time point HG, HG+LV-CTL, HG+LV-Sirt1, HG+LV CTL-RNAi, HG+LV-Sirt1-RNAi group CCK8 increased obviously (P 0.05).HG processing 24h. The levels of TGF- beta 1mRNA and protein were significantly up-regulated in group HG+LV-Sirt1 (P 0.05), and TGF- beta 1 mRNA and protein levels in group HG+LV-Sirt1 were significantly lower than that of HG, HG+LV-CTL, and TGF- beta 1 mRNA and protein levels in group LV-Sirt1-RNAi. There was no statistical difference. (3) high sugar increased STAT1 and STAT3 phosphorylation and acetylation level in a time dependent manner; (4) the level of STAT1 phosphorylation and acetylation of HG+LV-Sirt1-RNAi group increased in comparison with the NG+LV-CTL group, and the level of STAT1 acetylation in HG+LV-Sirt1 group decreased and HG+LV-Sirt1-RNAi group STAT1 compared with the HG+LV-CTL group. The level of acetylation increased (P 0.05), HG+LV-CTL group, HG+LV-Sirt1 group and HG+LV-Sirt1-RNAi group had no significant difference (P0.05). (5) positive, reverse immunoprecipitation can detect the combination of Sirt1 protein and STAT1 protein. Conclusion over expression of Sirt1 level can inhibit the proliferation of human glomerular mesangial cells induced by high glucose, TGF- beta 1 as well as the level of acetylation of STAT1 and STAT3, on the contrary, the expression of silent Sirt1 enhanced the treatment effect of high glucose; Sirt1 may play its role in antagonizing high glucose induced effect by deacetylation of STAT1; Sirt1 may be the target for the prevention and control of diabetic nephropathy.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.2;R692.9

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