本文選題：糖尿病腎病　切入點(diǎn)：Sirt1　出處：《鄭州大學(xué)》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(yán)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(diào)控,進(jìn)而通過不同的作用機(jī)制損害腎臟結(jié)構(gòu)及功能,最終加速疾病的進(jìn)展。作為腎臟濾過屏障最為重要的組分,足細(xì)胞受到炎性因子損害的現(xiàn)象日益受到關(guān)注。但糖尿病腎病炎癥發(fā)生機(jī)制復(fù)雜,急需探索新的治療方案來延緩糖尿病腎病的進(jìn)展。Sirt1(Sirtuins 1)是一種營養(yǎng)及代謝相關(guān)蛋白,具有調(diào)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(xì)胞的損傷關(guān)系密切,且能夠參與調(diào)控糖尿病腎病炎癥反應(yīng),但相關(guān)機(jī)制仍需進(jìn)一步探討。TTP(Tristetraprolin)是一種AREs(AU-rich elements)結(jié)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(jié)合,進(jìn)而發(fā)揮抗炎作用。TTP的蛋白量及與目的m RNA的結(jié)合能力主要受TTP的磷酸化水平影響。TTP具有多個(gè)磷酸化位點(diǎn),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(xì)胞活動中發(fā)揮關(guān)鍵作用,是細(xì)胞信號傳導(dǎo)的重要通路。多項(xiàng)研究表明,Sirt1可通過P38-MAPK信號通路參與調(diào)控炎癥反應(yīng)。而Sirt1是否能夠通過P38-MAPK信號通路來調(diào)控TTP的表達(dá),進(jìn)而參與糖尿病腎病的炎癥反應(yīng)、引起足細(xì)胞損傷,尚未有研究報(bào)道。本實(shí)驗(yàn)從細(xì)胞水平探究Sirt1在高糖誘導(dǎo)的足細(xì)胞炎癥反應(yīng)及損傷中發(fā)揮的作用及具體機(jī)制。方法體外貼壁培養(yǎng)條件永生化小鼠足細(xì)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(xì)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(xì)胞培養(yǎng)箱中分化培養(yǎng)。0.25%胰酶消化,按需傳代,待細(xì)胞分化成熟后,隨機(jī)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(zhuǎn)染對照組;(5)正常糖+Sirt1 siRNA轉(zhuǎn)染組;(6)正常糖+空白對照慢病毒組;(7)正常糖+過表達(dá)Sirt1慢病毒組;(8)高糖+空白對照慢病毒組;(9)高糖+過表達(dá)Sirt1慢病毒組。實(shí)驗(yàn)處理后36h、48h收集細(xì)胞,分別提取細(xì)胞總RNA和總蛋白,然后應(yīng)用q RT-PCR和Western blot在基因及蛋白水平上檢測Sirt1、P38-MAPK、TTP、足細(xì)胞標(biāo)記蛋白Nephrin、Podocin、損傷因子Desmin以及炎癥因子IL-6、IL-18的表達(dá)水平。結(jié)果1.高糖條件下足細(xì)胞中Sirt1、TTP、Nephrin及Podocin的m RNA和蛋白表達(dá)減少(P0.05),Desmin、IL-6及IL-18的m RNA和蛋白表達(dá)增多(均P0.05),P38-MAPK的表達(dá)無明顯變化(P0.05),而p-P38-MAPK(即磷酸化的P38-MAPK)的蛋白表達(dá)增多(P0.05)。2.細(xì)胞免疫熒光雙染色顯示,正常糖濃度及高糖條件下足細(xì)胞中P38-MAPK與TTP的表達(dá)均存在共定位現(xiàn)象。免疫共沉淀也驗(yàn)證了P38-MAPK蛋白能與TTP蛋白結(jié)合。3.利用siRNA下調(diào)Sirt1的表達(dá)后,和正常糖濃度組相比,足細(xì)胞中Sirt1、TTP、Nephrin及Podocin的m RNA和蛋白表達(dá)降低(P0.05),Desmin、IL-6及IL-18的m RNA和蛋白表達(dá)增多(均P0.05),P38-MAPK的表達(dá)無明顯變化(P0.05),而p-P38-MAPK的蛋白表達(dá)增多(P0.05)。4.利用慢病毒上調(diào)Sirt1的表達(dá)后,與各自未轉(zhuǎn)染慢病毒組相比,足細(xì)胞中Sirt1、TTP、Nephrin及Podocin的m RNA和蛋白表達(dá)增多(P0.05),Desmin、IL-6及IL-18的m RNA和蛋白表達(dá)減少(均P0.05),P38-MAPK的表達(dá)無明顯變化(P0.05),而p-P38-MAPK的蛋白表達(dá)減少(P0.05)。結(jié)論1.Sirt1的缺失能夠誘導(dǎo)足細(xì)胞炎癥反應(yīng)和損傷。2.上調(diào)Sirt1的表達(dá)后能夠緩解高糖誘導(dǎo)的足細(xì)胞炎癥反應(yīng)和損傷。3.高糖環(huán)境下Sirt1可能是通過調(diào)控足細(xì)胞中P38-MAPK的磷酸化水平進(jìn)而影響TTP的表達(dá),最終導(dǎo)致足細(xì)胞發(fā)生炎癥反應(yīng)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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