高糖环境下Sirt1通过P38-MAPK影响小鼠足细胞中TTP及炎症因子的表达

发布时间：2018-03-28 17:44

  本文选题：糖尿病肾病　切入点：Sirt1　出处：《郑州大学》2017年硕士论文

【摘要】：背景及目的糖尿病肾病(diabetic nephropathy,DN)作为糖尿病(diabetic mellitus,DM)最常见且最严重的并发症之一,在欧美国家中,DN是引起终末期肾脏病(end stage renal disease,ESRD)的首位病因。糖尿病肾病早期即可发生炎性因子分泌失调控,进而通过不同的作用机制损害肾脏结构及功能,最终加速疾病的进展。作为肾脏滤过屏障最为重要的组分,足细胞受到炎性因子损害的现象日益受到关注。但糖尿病肾病炎症发生机制复杂,急需探索新的治疗方案来延缓糖尿病肾病的进展。Sirt1(Sirtuins 1)是一种营养及代谢相关蛋白,具有调节表观遗传基因沉默、抑制r RNA重组等作用。有研究指出Sirt1与足细胞的损伤关系密切,且能够参与调控糖尿病肾病炎症反应,但相关机制仍需进一步探讨。TTP(Tristetraprolin)是一种AREs(AU-rich elements)结合蛋白,可直接与炎症因子TNF-α及IL-6的m RNA 3’UTR的AU区段结合,进而发挥抗炎作用。TTP的蛋白量及与目的m RNA的结合能力主要受TTP的磷酸化水平影响。TTP具有多个磷酸化位点,可被多种蛋白磷酸化。而P38-MAPK是一种可磷酸化TTP的重要酶,在多种细胞活动中发挥关键作用,是细胞信号传导的重要通路。多项研究表明,Sirt1可通过P38-MAPK信号通路参与调控炎症反应。而Sirt1是否能够通过P38-MAPK信号通路来调控TTP的表达,进而参与糖尿病肾病的炎症反应、引起足细胞损伤,尚未有研究报道。本实验从细胞水平探究Sirt1在高糖诱导的足细胞炎症反应及损伤中发挥的作用及具体机制。方法体外贴壁培养条件永生化小鼠足细胞(conditionally immortalized mouse podocyte,MPC),先将MPC置于含10%胎牛血清、5.6mmol/L葡萄糖和4ng/ml小鼠γ-干扰素的RPMI 1640培养基,33℃、5%CO2细胞培养箱中增殖培养;然后将MPC置于含10%胎牛血清、5.6mmol/L葡萄糖、不含小鼠γ-干扰素的RPMI1640培养基,37℃、5%CO2细胞培养箱中分化培养。0.25%胰酶消化,按需传代,待细胞分化成熟后,随机分为以下9组:(1)正常糖浓度组(含5.6mmol/L D-葡萄糖);(2)甘露醇组(含5.6mmol/L D-葡萄糖+19.4mmol/L D-甘露醇);(3)高糖浓度组(含25mmol/L D-葡萄糖);(4)正常糖+转染对照组;(5)正常糖+Sirt1 siRNA转染组;(6)正常糖+空白对照慢病毒组;(7)正常糖+过表达Sirt1慢病毒组;(8)高糖+空白对照慢病毒组;(9)高糖+过表达Sirt1慢病毒组。实验处理后36h、48h收集细胞,分别提取细胞总RNA和总蛋白,然后应用q RT-PCR和Western blot在基因及蛋白水平上检测Sirt1、P38-MAPK、TTP、足细胞标记蛋白Nephrin、Podocin、损伤因子Desmin以及炎症因子IL-6、IL-18的表达水平。结果1.高糖条件下足细胞中Sirt1、TTP、Nephrin及Podocin的m RNA和蛋白表达减少(P0.05),Desmin、IL-6及IL-18的m RNA和蛋白表达增多(均P0.05),P38-MAPK的表达无明显变化(P0.05),而p-P38-MAPK(即磷酸化的P38-MAPK)的蛋白表达增多(P0.05)。2.细胞免疫荧光双染色显示,正常糖浓度及高糖条件下足细胞中P38-MAPK与TTP的表达均存在共定位现象。免疫共沉淀也验证了P38-MAPK蛋白能与TTP蛋白结合。3.利用siRNA下调Sirt1的表达后,和正常糖浓度组相比,足细胞中Sirt1、TTP、Nephrin及Podocin的m RNA和蛋白表达降低(P0.05),Desmin、IL-6及IL-18的m RNA和蛋白表达增多(均P0.05),P38-MAPK的表达无明显变化(P0.05),而p-P38-MAPK的蛋白表达增多(P0.05)。4.利用慢病毒上调Sirt1的表达后,与各自未转染慢病毒组相比,足细胞中Sirt1、TTP、Nephrin及Podocin的m RNA和蛋白表达增多(P0.05),Desmin、IL-6及IL-18的m RNA和蛋白表达减少(均P0.05),P38-MAPK的表达无明显变化(P0.05),而p-P38-MAPK的蛋白表达减少(P0.05)。结论1.Sirt1的缺失能够诱导足细胞炎症反应和损伤。2.上调Sirt1的表达后能够缓解高糖诱导的足细胞炎症反应和损伤。3.高糖环境下Sirt1可能是通过调控足细胞中P38-MAPK的磷酸化水平进而影响TTP的表达,最终导致足细胞发生炎症反应和损伤。
[Abstract]:Background and objective: diabetic nephropathy (diabetic, nephropathy, DN) (diabetic mellitus, DM as diabetes) one of the most common complications and the most serious, in Europe and the United States, DN is the cause of end-stage renal disease (end stage renal disease, ESRD). The first cause of diabetic nephropathy occurs in the early stage of secretion of inflammatory cytokines dysregulation then, the structure and function of the damage mechanism of different kidney diseases. As the progress of the final acceleration of kidney filtration barrier is the most important component of podocyte by inflammatory cytokines is getting more and more attention to. But the inflammation of diabetic nephropathy has a complex mechanism, progress of.Sirt1 urgently need to explore new treatments to delay diabetic nephropathy the (Sirtuins 1) is a kind of nutrition and metabolism related protein, can regulate epigenetic gene silencing and inhibition of R RNA recombination. Studies have indicated that Sirt1 and podocyte injury Closely related, and can participate in the regulation of inflammatory reaction of diabetic nephropathy, but the mechanisms still need further discussion of.TTP (Tristetraprolin) is a kind of AREs (AU-rich elements) binding protein, AU segment m RNA directly with the inflammatory factor TNF- alpha and IL-6 UTR in 3 "combination, and play the anti-inflammatory effects of.TTP protein binding capacity the purpose of M and RNA is mainly affected by the phosphorylation of TTP.TTP with multiple phosphorylation sites, can be a variety of protein phosphorylation. P38-MAPK is an important enzyme of TTP phosphorylation, play a key role in a variety of cellular activities, is an important pathway of signal transduction in cells. Many studies show that Sirt1 through the P38-MAPK signaling pathway involved in the regulation of inflammation. And whether can Sirt1 through P38-MAPK signaling pathway to regulate the expression of TTP, and is involved in the inflammatory response in diabetic nephropathy, cell damage caused by foot, not yet Studies have been reported. This experiment explore Sirt1 play in podocyte inflammation and injury induced by high glucose and the role of specific mechanisms at the cellular level. Methods in vitro adherent culture conditions of immortalized mouse podocytes (conditionally immortalized mouse podocyte, MPC), first placed MPC with 10% fetal bovine serum, 5.6mmol/L glucose and 4ng/ml mouse IFN RPMI 1640 medium, 33 C, 5%CO2 cells proliferation box; then MPC in 5.6mmol/L containing 10% fetal bovine serum, glucose, free mouse gamma interferon RPMI1640 medium, 37 C, box 5%CO2 trypsin digestion in differentiated.0.25% cells, according to the passage, for cell differentiation, were randomly divided into 9 groups: (1) normal glucose concentration group (5.6mmol/L containing D- glucose); (2) the mannitol group (containing 5.6mmol/L D- glucose +19.4mmol/L D- mannitol); (3) high glucose group (25mmol/L containing D- glucose 钀勭硸);(4)姝ｅ父绯，

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