本文選題：腎臟纖維化 + 轉(zhuǎn)錄生長因子β�。� 參考：《南京大學(xué)》2017年博士論文

【摘要】：慢性腎臟病(Chronic kidney disease,CKD)患病率和病死率居高不下己成為全球范圍內(nèi)的重大公共健康問題,造成了巨大的經(jīng)濟(jì)損失。腎臟纖維化是包括糖尿病腎病、高血壓腎病、IgA腎病等在內(nèi)的多種CKD的共同病理特征。腎臟纖維化過程中,αα-平滑肌肌動蛋白(Alpha smooth musle actin,α-SMA)陽性表達(dá)的肌成纖維細(xì)胞增殖能力增強(qiáng),細(xì)胞外基質(zhì)(Extracellular matrix,ECM)累積,形成永久的疤痕組織,腎臟功能下降。深入研究腎臟纖維化的分子機(jī)制,從根源出發(fā)尋找積極、安全、有效緩解腎臟纖維化的干預(yù)手段是我們急需面對的問題。Klotho是一個腎臟中特異性高表達(dá)的新型抗衰老蛋白,近年來研究發(fā)現(xiàn)Klotho有腎臟保護(hù)和抗纖維化的作用。Klotho蛋白分為膜型和分泌型,膜型Klotho主要作為FGF23等的共受體發(fā)揮作用;分泌型Klotho分布在循環(huán)系統(tǒng)中起激素調(diào)節(jié)作用。在腎臟纖維化過程中,氧化應(yīng)激和炎癥通路、TGFβ信號通路和Wnt/β-catenin信號通路共同調(diào)控促纖維化過程。近年來研究表明Klotho蛋白能夠抑制炎癥反應(yīng)、氧化應(yīng)激、TGFβ信號通路和Wnt/β-catenin信號通路。在腎臟纖維化相關(guān)疾病患者和動物模型中,Klotho在疾病進(jìn)程早期即出現(xiàn)一致的表達(dá)下調(diào)并與疾病進(jìn)程密切相關(guān),外源性補(bǔ)充Klotho能夠有效抵抗腎臟纖維化。綜上,Klotho不僅是腎臟疾病早期的診斷標(biāo)志物還是治療纖維化相關(guān)腎臟疾病的有效靶點(diǎn)。在腎臟纖維化過程中Klotho下調(diào)的機(jī)制還未完全明確,Klotho基因啟動子上含有密集的CpG位點(diǎn),易受甲基化調(diào)控。己證明DNA甲基化調(diào)控Klotho的組織特異性表達(dá),表觀遺傳調(diào)控在腎臟纖維化進(jìn)程也發(fā)揮重要作用。種種線索提示在腎臟纖維化過程中Klotho表達(dá)可能受表觀遺傳DNA甲基化調(diào)控。我們構(gòu)建了兩個腎臟纖維化小鼠模型:單側(cè)輸尿管結(jié)扎(UUO)和腺嘌呤(Adenine)模型,檢測腎臟組織中Klotho的表達(dá)和甲基化情況,結(jié)果證明在腎臟纖維化過程中Klotho表達(dá)下降且受甲基化調(diào)控。DNA甲基化過程主要由DNA甲基轉(zhuǎn)移酶(DNAmethyltransferase,DNMT)將S-腺苷-甲硫氨酸(S-adenosyl methionine,SAM)上的甲基基團(tuán)轉(zhuǎn)移到胞嘧陡上,引起基因表達(dá)抑制。在哺乳動物中DNMT主要有DNMT1、DNMT3a和DNMT3b三種亞型。接下來我們進(jìn)一步探究造成Klotho啟動子甲基化的上游事件,我們進(jìn)一步檢測了腎臟組織中DNMT表達(dá),結(jié)果發(fā)現(xiàn)纖維化過程中DNMT1和DNMT3a表達(dá)增加。研究發(fā)現(xiàn)TGFβ有抑制Klotho表達(dá)和調(diào)控甲基化的雙效作用,可能是調(diào)控腎臟纖維化過程中Klotho啟動子甲基化抑制的關(guān)鍵因子。我們的實(shí)驗(yàn)結(jié)果證明纖維化腎臟組織中TGFβ表達(dá)急劇增加,且TGFβ單獨(dú)處理能抑制腎臟上皮細(xì)胞中Klotho的表達(dá),促進(jìn)Klotho啟動子甲基化,并能促進(jìn)DNMT1和DNMT3a的表達(dá),引起和纖維化腎臟中一致的變化。通過網(wǎng)站分析預(yù)測與DNMT1和DNMT3a評分較高的兩種miRNA,miR-152和miR-30a。在纖維化的腎臟組織和TGFβ誘導(dǎo)的腎臟上皮細(xì)胞中miR-152和miR-30a表達(dá)減少,抑制TGFβ Ⅰ型受體后,miR-152和miR-30a的表達(dá)抑制作用減弱。過表達(dá)miR-152和miR-30a能夠分別抑制DNMT1和DNMT3a的表達(dá)。證明在腎臟纖維化過程中TGFβ通過抑制miR-152和miR-30a增加DNMT1和DNMT3a的表達(dá)量。采用小發(fā)卡RNA分別干擾DNMT1和DNMT3a表達(dá)的研究結(jié)果證明Klotho的啟動子甲基化過程由DNMT1和DNMT3a共同調(diào)控。為了驗(yàn)證TGFβ和DNA甲基化的作用,我們用TGFβ Ⅰ型受體抑制劑SB431542和DNMT抑制劑地西他濱(5-氮雜-2'-脫氧胞嘧啶核苷,5-Aza-2'-deoxycytidine,5Aza)進(jìn)行體內(nèi)實(shí)驗(yàn)。SB431542能夠有效緩解腎臟纖維化并抑制DNMT1和DNMT3a的表達(dá)上調(diào),還能夠減弱Klotho啟動子甲基化水平并恢復(fù)其表達(dá),證明了 TGFβ的關(guān)鍵誘導(dǎo)作用。5Aza能夠有效恢復(fù)Klotho的表達(dá)抑制其甲基化并有效地緩解腎臟纖維化。在小鼠體內(nèi)干擾Klotho表達(dá)后,發(fā)現(xiàn)5Aza緩解腎臟纖維化的作用受到抑制,證明Klotho是5Aza抗纖維化作用的關(guān)鍵靶點(diǎn)。綜上所述,本研究證實(shí)了在腎臟纖維化過程中Klotho的表達(dá)抑制受啟動子甲基化調(diào)控。并證實(shí)了在腎臟纖維化過程中Klotho甲基化的上游事件。我們通過體內(nèi)實(shí)驗(yàn)和體外實(shí)驗(yàn)證明TGFβ是誘導(dǎo)腎臟纖維化中表觀調(diào)控的關(guān)鍵病理因素,TGFβ能夠抑制miR-152和miR-30a分別上調(diào)DNMT1和DNMT3a進(jìn)而促進(jìn)Klotho啟動子的異常高甲基化和Klotho蛋白的表達(dá)抑制。這些變化使Klotho的腎臟保護(hù)作用減弱加劇腎臟纖維化。我們的研究證明干預(yù)miRNA-DNMT-Klotho通路可能是治療纖維化相關(guān)腎臟疾病的積極有效的新方向。
[Abstract]:The prevalence of chronic kidney disease (Chronic kidney disease, CKD) and high mortality have become a major public health problem worldwide, causing huge economic losses. Renal fibrosis is a common pathological feature of various CKD, including diabetic nephropathy, hypertensive nephropathy, IgA nephropathy, and so on. In the process of renal fibrosis, alpha alpha - alpha - The proliferation ability of myofibroblast (Alpha smooth musle actin, alpha -SMA) positive expression of myofibroblast increased, the extracellular matrix (Extracellular matrix, ECM) accumulated, formed permanent scar tissue, and the renal function decreased. The molecular mechanism of renal fibrosis was studied in depth, to find active, safe and effective kidneys from the origin. The intervention of fibrosis is an urgent problem we need to face..Klotho is a novel antiaging protein with high specific expression in the kidney. In recent years, the study found that Klotho has renal protection and anti fibrosis effect,.Klotho protein is divided into membrane type and secretory type. Membrane type Klotho is mainly used as FGF23 and other common receptors; secretory Klotho During the process of renal fibrosis, oxidative stress and inflammatory pathways, TGF beta signaling pathway and Wnt/ beta -catenin signaling pathway co regulate the process of fibrosis during renal fibrosis. In recent years, studies have shown that Klotho protein can inhibit inflammatory response, oxidative stress, TGF beta signaling pathway and Wnt/ beta -catenin signaling pathway. In patients with renal fibrosis related diseases and animal models, Klotho has a consistent down-regulation in the early stages of the disease and is closely related to the process of disease. Exogenous Klotho can effectively resist renal fibrosis. To sum up, Klotho is not only a diagnostic marker for early renal disease, but also for the treatment of fibrosis related renal diseases. Effective targets. The mechanism of Klotho downregulation in the process of renal fibrosis is not completely clear. The Klotho gene promoter contains dense CpG loci, which is easily regulated by methylation. It has been proved that DNA methylation regulates the specific expression of Klotho, and epigenetic regulation plays an important role in the progression of renal fibrosis. Various clues suggest that the kidney is in the kidney. The expression of Klotho may be regulated by epigenetic DNA methylation during the process of filthy fibrosis. We constructed two mice models of renal fibrosis: unilateral ureteral ligation (UUO) and adenine (Adenine) model to detect the expression and methylation of Klotho in renal tissue. The results showed that the expression of Klotho in renal fibrosis was decreased and methylated in the process of renal fibrosis. The process of.DNA methylation is mainly controlled by DNA methyltransferase (DNAmethyltransferase, DNMT) to transfer the methyl group of S- adenosine methionine (S-adenosyl methionine, SAM) to cytosimon steepness, causing inhibition of gene expression. In mammals DNMT mainly consists of DNMT1, DNMT3a and DNMT3b three subtypes. In the upstream event of Klotho promoter methylation, we further detected the expression of DNMT in the renal tissue, and found that the expression of DNMT1 and DNMT3a increased during the process of fibrosis. The study found that TGF beta has the double effect of inhibiting the expression of Klotho and regulating methylation, which may be the regulation of the inhibition of the methylation of Klotho promoter in the process of renal fibrosis. Key factors. Our experimental results show that the expression of TGF beta in the renal fibrotic tissue increases sharply, and the TGF beta alone can inhibit the expression of Klotho in the renal epithelial cells, promote the methylation of the Klotho promoter, and promote the expression of DNMT1 and DNMT3a, and cause the same changes in the renal fibrosis. The two kinds of miRNA with higher T3a score, miR-152 and miR-30a. were reduced in the renal tissue of fibrosis and the expression of miR-152 and miR-30a in the renal epithelial cells induced by TGF beta. The inhibition of the expression of miR-152 and miR-30a was weakened after the inhibition of TGF beta type I receptor. In the process of fibrosis, TGF beta increases the expression of DNMT1 and DNMT3a by inhibiting miR-152 and miR-30a. The results of DNMT1 and DNMT3a expression using small hairpin RNA show that the promoter methylation process of Klotho is regulated by DNMT1 and DNMT3a. The experimental.SB431542 in 431542 and DNMT inhibitors (5- -2'- deoxycytidine nucleoside, 5-Aza-2'-deoxycytidine, 5Aza) can effectively alleviate renal fibrosis and inhibit the up-regulated expression of DNMT1 and DNMT3a, and also reduce the level of Klotho promoter methylation and restore its expression, which proves that the key inducement of TGF beta is.5A. Za can effectively restore the expression of Klotho to inhibit its methylation and effectively alleviate renal fibrosis. After interfering with Klotho expression in mice, it is found that the role of 5Aza in alleviating renal fibrosis is inhibited, which proves that Klotho is the key target of the anti fibrosis effect of 5Aza. To sum up, this research confirms Klotho in the process of renal fibrosis. The expression inhibits the regulation of promoter methylation and confirms the upstream event of Klotho methylation during renal fibrosis. Through in vivo and in vitro experiments, we have demonstrated that TGF beta is a key pathological factor in the apparent regulation of renal fibrosis. TGF beta can inhibit miR-152 and miR-30a up to DNMT1 and DNMT3a and then promote Klotho, respectively. The abnormal hypermethylation of the promoter and the inhibition of the expression of Klotho protein. These changes weaken the renal protective effect of Klotho and aggravate the renal fibrosis. Our study has shown that intervention in the miRNA-DNMT-Klotho pathway may be a positive and effective new direction for the treatment of fibrosis related renal diseases.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R692

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