本文選題：糖尿病腎病　切入點(diǎn)：血管緊張素Ⅱ　出處：《第二軍醫(yī)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景及目的糖尿病腎病是導(dǎo)致終末期腎病的最主要病因,超40%的腎臟替代治療患者是由糖尿病引起。臨床主要表現(xiàn)為大量蛋白尿。研究認(rèn)為蛋白尿的產(chǎn)生與腎小球足細(xì)胞損傷密切相關(guān)。在糖尿病腎病中,有多種因素如高血糖、血管緊張素Ⅱ(Ang-Ⅱ)等導(dǎo)致足細(xì)胞凋亡。嚴(yán)格控制血糖及應(yīng)用腎素-血管緊張素系統(tǒng)(RAS)阻斷劑能夠明顯減少尿蛋白的產(chǎn)生。SIRT1是一種沉默信息調(diào)節(jié)因子,具有NAD+依賴的去乙�；富钚�,參與了多種疾病的發(fā)病過(guò)程,其中也包括糖尿病腎病。在糖尿病腎病中,SIRT1參與了炎癥、細(xì)胞凋亡、纖維化等多個(gè)病理過(guò)程。在細(xì)胞凋亡方面,既往研究發(fā)現(xiàn),高糖和Ang-Ⅱ可以通過(guò)不同的信號(hào)通路影響SIRT1的表達(dá)及活性,Ang-Ⅱ誘導(dǎo)內(nèi)皮細(xì)胞凋亡并激活了P38/SIRT1通路。高糖培養(yǎng)可以激活足細(xì)胞中的AMPK/SIRT1通路介導(dǎo)細(xì)胞凋亡。在臨床應(yīng)用中,奧美沙坦能夠減少糖尿病腎病患者尿蛋白,但是在血糖控制不佳的患者中,奧美沙坦降低尿蛋白的作用并不明顯。說(shuō)明高糖狀態(tài)下奧美沙坦的治療會(huì)受到影響。也提示高糖和Ang-Ⅱ可能通過(guò)不同通路誘導(dǎo)了足細(xì)胞凋亡。因此我們?cè)O(shè)想,在糖尿病腎病中,Ang-Ⅱ和高糖可能通過(guò)不同的信號(hào)通路(P38/SIRT1通路與AMPK/SIRT1通路)影響SIRT1的表達(dá),從而導(dǎo)致了足細(xì)胞的凋亡,而奧美沙坦只能對(duì)P38/SIRT1通路起作用。奧美沙坦是臨床常用的血管緊張素Ⅱ抑制劑,能夠抑制Ang-Ⅱ與血管緊張素Ⅱ受體的結(jié)合。既往認(rèn)為Ang-Ⅱ主要通過(guò)絲裂原活化蛋白激酶(MAPK),尤其是ERK1/2信號(hào)通路誘導(dǎo)足細(xì)胞凋亡。而Ang-Ⅱ是否也能通過(guò)P38/SIRT1通路介導(dǎo)足細(xì)胞凋亡目前缺乏相關(guān)報(bào)道。研究方法1.動(dòng)物實(shí)驗(yàn):糖尿病腎病模型db/db小鼠為糖尿病組,并分成兩組:安慰劑組(db/db+CMC)和奧美沙坦組(db/db+OM),C57BL小鼠作用為非糖尿病組(WT),每組10只。在小鼠10-12周齡時(shí),開(kāi)始藥物灌胃處理,安慰劑為0.5%羧甲基纖維素鈉(CMC),奧美沙坦劑量為每天20mg/kg。在處理的第0、2、4、6、8、10、12周測(cè)尾靜脈血糖并收集24小時(shí)尿液測(cè)尿蛋白。第12周處死動(dòng)物,留取腎組織做病理切片及提取組織蛋白,通過(guò)western blot分析蛋白通路變化。2.細(xì)胞實(shí)驗(yàn):在33℃條件下,條件永生化小鼠足細(xì)胞可以進(jìn)行傳代培養(yǎng),并且需要在培養(yǎng)基(RPMI1640)中添加10%牛血清和1×104U/L的γ干擾素;在37℃條件下細(xì)胞開(kāi)始分化,分化完成需要14天,分化時(shí)培養(yǎng)基中不加γ干擾素。分化完成后給予葡萄糖、Ang-Ⅱ、奧美沙坦等刺激處理,收集細(xì)胞用流式細(xì)胞儀檢測(cè)凋亡或提取蛋白用WesternBlot方法分析通路變化。3.統(tǒng)計(jì)學(xué)處理:所有數(shù)據(jù)結(jié)果用均數(shù)±標(biāo)準(zhǔn)差(±s)表示,利用Graphpad Prism5.0軟件進(jìn)行數(shù)據(jù)統(tǒng)計(jì)分析和制圖。組間的差異分析方法采用one wayANOVA,定義P值0.05為差異有統(tǒng)計(jì)學(xué)意義。研究結(jié)果1.奧美沙坦治療12周后可以減少db/db小鼠的尿蛋白,而不影響小鼠的體重和血糖。在db/db小鼠腎臟組織中,奧美沙坦明顯抑制p38/SIRT1通路,減少了足細(xì)胞的凋亡,但對(duì)AMPK沒(méi)有影響。2.奧美沙坦能部分抑制高糖激活的AMPK/SIRT1通路,減少足細(xì)胞凋亡。3.Ang-Ⅱ激活p38/SIRT1通路誘導(dǎo)足細(xì)胞凋亡,而奧美沙坦明顯的抑制這條通路,保護(hù)足細(xì)胞;P38抑制劑s8307具有類似作用。結(jié)論本課題驗(yàn)證了SIRT1在糖尿病腎病足細(xì)胞中具有重要的保護(hù)作用。高糖通過(guò)AMPK途徑抑制SIRT1活性誘導(dǎo)足細(xì)胞凋亡;血管緊張素II通過(guò)p38/SIRT1通路觸發(fā)了足細(xì)胞的凋亡。奧美沙坦能夠抑制Ang-Ⅱ/p38/SIRT1通路,減少足細(xì)胞凋亡,降低糖尿病腎病小鼠尿蛋白;P38抑制劑s8307具有類似作用。
[Abstract]:Background and objective: diabetic nephropathy is the main cause leading to end stage renal disease, over 40% of the renal replacement therapy patients is caused by diabetes. The main clinical manifestations of proteinuria. Studies suggest that podocyte injury and proteinuria are closely related. In diabetic nephropathy, there are many factors such as high blood glucose, blood pressure angiotensin II (Ang- II) as a result of podocyte apoptosis. Strict blood glucose control and application of the renin-angiotensin system (RAS) blocking agent can significantly reduce the production of.SIRT1 urine protein is a kind of silent information regulator, has NAD+ dependent deacetylase activity is involved in the pathogenesis of many diseases, including including diabetic nephropathy. In diabetic nephropathy, SIRT1 is involved in inflammation, apoptosis, fibrosis and other multiple pathological processes. In apoptosis, previous studies have found that high glucose and Ang- II The expression and activity of different signaling pathways affect SIRT1, Ang- II induced endothelial cell apoptosis and activation of the P38/SIRT1 pathway. High glucose can activate AMPK/SIRT1 pathway in podocytes apoptosis. In clinical application, olmesartan can reduce urinary protein in diabetic nephropathy patients, but in patients with poor glycemic control. Olmesartan reduces urinary protein, the effect is not obvious. The treatment under high glucose condition olmesartan will be affected. Also suggested that high glucose and Ang- could be induced by different pathways of podocyte apoptosis. So we suppose, in diabetic nephropathy, Ang- II and glucose may through different signaling pathways (P38/SIRT1 pathway with the AMPK/SIRT1 pathway) affect the expression of SIRT1, thus leading to podocyte apoptosis, and olmesartan can only work on P38/SIRT1 pathway. Olmesartan is commonly used in clinical blood Angiotensin II inhibitor, can inhibit Ang- II and angiotensin II receptor binding. Previous studies showed that Ang- II mainly through mitogen activated protein kinase (MAPK), especially induced podocyte apoptosis and ERK1/2 signaling pathway. Ang- II can the current lack of relevant reports of apoptosis mediated by P38/SIRT1 signaling in podocytes. Research methods 1. animal experiment: db/db mice model of diabetic nephropathy in diabetic group, and divided into two groups: placebo group (db/db+CMC) and olmesartan group (db/db+OM), C57BL mice as non diabetes group (WT), 10 rats in each group. The mice at the age of 10-12 weeks, starting drug treatment by gavage, placebo was 0.5% cm sodium carboxymethyl cellulose (CMC), olmesartan dose was 20mg/kg. per day in the treatment of tail vein blood glucose measurement in 0,2,4,6,8,10,12 weeks and collected 24 hours urine test urine protein. Twelfth weeks animal, nephridial tissue pathological slices and Extraction of tissue protein, through the western analysis of blot protein signaling pathway in.2. cells: experiment under the condition of 33 DEG C, conditionally immortalized mouse podocytes can be passaged, and in the culture medium (RPMI1640) supplemented with 10% bovine serum and 1 x 104U/L interferon gamma; under the condition of 37 DEG C cells differentiation, differentiation 14 days, the differentiation culture medium without interferon gamma. After differentiation to glucose, Ang- II, olmesartan stimulation treatment, cells were collected through the analysis of path changes statistically.3. WesternBlot method with flow cytometry was used to detect the apoptosis or protein extraction: all the data with the standard deviation (SD s) said, statistical data analysis and mapping using Graphpad Prism5.0 software. Differences between groups were analyzed by one wayANOVA method, the definition of the P value is 0.05. There was a significant difference on the results of the 1. olmesartan 12 After a week can reduce urinary protein in db/db mice, and the mice did not affect body weight and blood glucose. In the kidneys of db/db mice, olmesartan inhibited p38/SIRT1 pathway, reduce podocyte apoptosis, but did not affect.2. AMPK/SIRT1 pathway of olmesartan could partially inhibit high glucose on activation of AMPK, reduce the apoptosis of podocyte.3.Ang- II induced podocyte apoptosis and activation of the p38/SIRT1 pathway, and olmesartan significantly inhibit this pathway, protect the podocyte; P38 inhibitor s8307 has similar effect. Conclusion the study proved that SIRT1 has an important protective role in diabetic nephropathy in podocytes. High glucose induced podocyte apoptosis by AMPK pathway inhibits SIRT1 activity; angiotensin II through the p38/SIRT1 pathway triggered podocyte apoptosis. Olmesartan can inhibit Ang- II /p38/SIRT1 pathway, reduce podocyte apoptosis, reduce diabetic nephropathy rat urine and small eggs White; P38 inhibitor s8307 has a similar effect.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.2;R692.9

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