本文選題：糖尿病腎病　切入點：Sirt1　出處：《鄭州大學》2017年碩士論文

【摘要】：背景及目的糖尿病腎病(diabetic nephropathy,DN)作為糖尿病(diabetic mellitus,DM)最常見且最嚴重的并發(fā)癥之一,在歐美國家中,DN是引起終末期腎臟病(end stage renal disease,ESRD)的首位病因。糖尿病腎病早期即可發(fā)生炎性因子分泌失調控,進而通過不同的作用機制損害腎臟結構及功能,最終加速疾病的進展。作為腎臟濾過屏障最為重要的組分,足細胞受到炎性因子損害的現象日益受到關注。但糖尿病腎病炎癥發(fā)生機制復雜,急需探索新的治療方案來延緩糖尿病腎病的進展。Sirt1(Sirtuins 1)是一種營養(yǎng)及代謝相關蛋白,具有調節(jié)表觀遺傳基因沉默、抑制r RNA重組等作用。有研究指出Sirt1與足細胞的損傷關系密切,且能夠參與調控糖尿病腎病炎癥反應,但相關機制仍需進一步探討。TTP(Tristetraprolin)是一種AREs(AU-rich elements)結合蛋白,可直接與炎癥因子TNF-α及IL-6的m RNA 3’UTR的AU區(qū)段結合,進而發(fā)揮抗炎作用。TTP的蛋白量及與目的m RNA的結合能力主要受TTP的磷酸化水平影響。TTP具有多個磷酸化位點,可被多種蛋白磷酸化。而P38-MAPK是一種可磷酸化TTP的重要酶,在多種細胞活動中發(fā)揮關鍵作用,是細胞信號傳導的重要通路。多項研究表明,Sirt1可通過P38-MAPK信號通路參與調控炎癥反應。而Sirt1是否能夠通過P38-MAPK信號通路來調控TTP的表達,進而參與糖尿病腎病的炎癥反應、引起足細胞損傷,尚未有研究報道。本實驗從細胞水平探究Sirt1在高糖誘導的足細胞炎癥反應及損傷中發(fā)揮的作用及具體機制。方法體外貼壁培養(yǎng)條件永生化小鼠足細胞(conditionally immortalized mouse podocyte,MPC),先將MPC置于含10%胎牛血清、5.6mmol/L葡萄糖和4ng/ml小鼠γ-干擾素的RPMI 1640培養(yǎng)基,33℃、5%CO2細胞培養(yǎng)箱中增殖培養(yǎng);然后將MPC置于含10%胎牛血清、5.6mmol/L葡萄糖、不含小鼠γ-干擾素的RPMI1640培養(yǎng)基,37℃、5%CO2細胞培養(yǎng)箱中分化培養(yǎng)。0.25%胰酶消化,按需傳代,待細胞分化成熟后,隨機分為以下9組:(1)正常糖濃度組(含5.6mmol/L D-葡萄糖);(2)甘露醇組(含5.6mmol/L D-葡萄糖+19.4mmol/L D-甘露醇);(3)高糖濃度組(含25mmol/L D-葡萄糖);(4)正常糖+轉染對照組;(5)正常糖+Sirt1 siRNA轉染組;(6)正常糖+空白對照慢病毒組;(7)正常糖+過表達Sirt1慢病毒組;(8)高糖+空白對照慢病毒組;(9)高糖+過表達Sirt1慢病毒組。實驗處理后36h、48h收集細胞,分別提取細胞總RNA和總蛋白,然后應用q RT-PCR和Western blot在基因及蛋白水平上檢測Sirt1、P38-MAPK、TTP、足細胞標記蛋白Nephrin、Podocin、損傷因子Desmin以及炎癥因子IL-6、IL-18的表達水平。結果1.高糖條件下足細胞中Sirt1、TTP、Nephrin及Podocin的m RNA和蛋白表達減少(P0.05),Desmin、IL-6及IL-18的m RNA和蛋白表達增多(均P0.05),P38-MAPK的表達無明顯變化(P0.05),而p-P38-MAPK(即磷酸化的P38-MAPK)的蛋白表達增多(P0.05)。2.細胞免疫熒光雙染色顯示,正常糖濃度及高糖條件下足細胞中P38-MAPK與TTP的表達均存在共定位現象。免疫共沉淀也驗證了P38-MAPK蛋白能與TTP蛋白結合。3.利用siRNA下調Sirt1的表達后,和正常糖濃度組相比,足細胞中Sirt1、TTP、Nephrin及Podocin的m RNA和蛋白表達降低(P0.05),Desmin、IL-6及IL-18的m RNA和蛋白表達增多(均P0.05),P38-MAPK的表達無明顯變化(P0.05),而p-P38-MAPK的蛋白表達增多(P0.05)。4.利用慢病毒上調Sirt1的表達后,與各自未轉染慢病毒組相比,足細胞中Sirt1、TTP、Nephrin及Podocin的m RNA和蛋白表達增多(P0.05),Desmin、IL-6及IL-18的m RNA和蛋白表達減少(均P0.05),P38-MAPK的表達無明顯變化(P0.05),而p-P38-MAPK的蛋白表達減少(P0.05)。結論1.Sirt1的缺失能夠誘導足細胞炎癥反應和損傷。2.上調Sirt1的表達后能夠緩解高糖誘導的足細胞炎癥反應和損傷。3.高糖環(huán)境下Sirt1可能是通過調控足細胞中P38-MAPK的磷酸化水平進而影響TTP的表達,最終導致足細胞發(fā)生炎癥反應和損傷。
[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父緋，

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