本文關(guān)鍵詞：激活FXR下調(diào)visfatin對(duì)糖尿病腎病的作用及機(jī)制研究　出處：《第三軍醫(yī)大學(xué)》2017年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 法尼酯X受體 visfatin 糖尿病腎病 系膜細(xì)胞

【摘要】：背景和目的:糖尿病腎病(Diabetic Nephropathy,DN)是糖尿病的重要并發(fā)癥,也是最常見(jiàn)的導(dǎo)致終末期腎臟疾病(End-stage renal disease,ESRD)的病因之一。其主要病理特征為腎臟結(jié)構(gòu)改變包括腎小球肥大、系膜基質(zhì)增厚、腎小球足細(xì)胞損傷,導(dǎo)致腎小球硬化和腎小管間質(zhì)纖維化。這些變化共同導(dǎo)致了進(jìn)行性蛋白尿增多和腎小球?yàn)V過(guò)率減少,最后發(fā)展為終末期腎病。糖尿病腎病的發(fā)病機(jī)制目前并不完全清楚,研究表明高血壓、高血糖、晚期糖基化終末產(chǎn)物、脂質(zhì)代謝異常、脂肪堆積、促纖維化生長(zhǎng)因子、炎癥因子、氧化應(yīng)激等在糖尿病腎病進(jìn)展中發(fā)揮一定的作用。法尼酯X受體(Farnesoid X receptor,FXR)屬于核受體超家族成員,是一種細(xì)胞內(nèi)信號(hào)蛋白,作為多功能轉(zhuǎn)錄調(diào)節(jié)因子激活或抑制靶基因表達(dá)。FXR在肝臟、腸、腎上腺和腎臟等組織都有高表達(dá)。現(xiàn)有研究表明FXR在糖尿病腎病發(fā)生發(fā)展中發(fā)揮重要作用,可能與FXR參與調(diào)控糖脂代謝基因表達(dá)有關(guān),但具體調(diào)控機(jī)制仍不清楚。visfatin是新近分離并鑒定的一種分泌型蛋白,亦稱(chēng)前B細(xì)胞克隆增強(qiáng)因子(pre-B cell colony ehancing factor,PBEF),在脂肪組織、肝臟、心臟、骨骼肌、腦、腎臟等組織中均有表達(dá)。研究發(fā)現(xiàn)visfatin在糖脂代謝、胰島素抵抗、細(xì)胞增殖、分化、凋亡和炎癥反應(yīng)等過(guò)程中發(fā)揮重要作用。研究發(fā)現(xiàn)visfatin在糖尿病患者血清濃度明顯升高,參與了調(diào)控炎癥因子表達(dá)、內(nèi)皮功能損傷、胰島素抵抗及動(dòng)脈粥樣硬化等。體外研究亦發(fā)現(xiàn)visfatin在高糖誘導(dǎo)腎小球系膜細(xì)胞及腎小管上皮細(xì)胞表達(dá)顯著升高,外源性visfatin可促進(jìn)腎小球系膜細(xì)胞葡萄糖轉(zhuǎn)運(yùn)蛋白GLUT-1的表達(dá)上調(diào),促進(jìn)葡萄糖吸收,并刺激促纖維化基因表達(dá)。我們前期研究發(fā)現(xiàn)血清visfatin水平在糖尿病腎病患者明顯升高,且與炎癥因子呈正相關(guān),與腎功能水平呈顯著負(fù)相關(guān),提示visfatin很可能與糖尿病腎病的進(jìn)程及炎癥狀態(tài)有密切相關(guān)。本研究擬通過(guò)體內(nèi)體外實(shí)驗(yàn)闡明FXR調(diào)控visfatin表達(dá)的分子機(jī)制及其在糖尿病腎病進(jìn)展過(guò)程中的作用,為深入認(rèn)識(shí)糖尿病腎病發(fā)病機(jī)制提供新的思路。方法:1.臨床研究部分:收集2014年6月-2015年12月我科收治經(jīng)腎活檢確診的2型糖尿病腎病患者的腎活檢標(biāo)本及臨床資料,并根據(jù)病理分期分為早期DN組13例、中期DN組16例、晚期DN組18例;收集我院泌尿外科同期收治的10例腎腫瘤手術(shù)患者遠(yuǎn)離腫瘤組織的正常組織標(biāo)本為對(duì)照組;對(duì)收集的腎組織標(biāo)本行PAS及visfatin免疫組化染色;患者臨床資料收集包括年齡、性別、24小時(shí)尿白蛋白、血肌酐(Scr)、血尿素氮(BUN),并根據(jù)Cockcroft-Grault公式計(jì)算腎小球?yàn)V過(guò)率(e GFR)。并對(duì)分析統(tǒng)計(jì)結(jié)果進(jìn)行如下比較:(1)比較各組24小時(shí)尿白蛋白、Scr、BUN、e GFR及visfatin表達(dá)半定量結(jié)果;(2)選取各期DN組患者24小時(shí)尿蛋白、Scr、BUN、e GFR結(jié)果,與visfatin表達(dá)半定量結(jié)果進(jìn)行相關(guān)性分析。2.細(xì)胞實(shí)驗(yàn)部分:(1)采用FXR激動(dòng)劑GW4064、FXR拮抗劑Guggulsterone處理人腎小球系膜細(xì)胞HMC,Real-time PCR和Western blot檢測(cè)visfatin的m RNA及蛋白表達(dá)變化;(2)分別給予高糖(HG)誘導(dǎo)HMC轉(zhuǎn)染si RNA沉默visfatin、GW4064或外源性visfatin處理:(1)Real-time PCR檢測(cè)visfatin的m RNA表達(dá),Western blot檢測(cè)visfatin、NF-κB、IκBα的蛋白表達(dá)水平,ELISA法檢測(cè)細(xì)胞上清MCP-1濃度;(2)Real-time PCR檢測(cè)TGF-β1、α-SMA、Collagen IV及FN的的m RNA表達(dá)水平,Western blot檢測(cè)TGF-β1、smad2/3、p-smad2/3、α-SMA、Collagen IV及FN的蛋白表達(dá)水平;(3)cck-8法檢測(cè)系膜細(xì)胞增殖,Real-time PCR和Western blot檢測(cè)增殖相關(guān)基因PCNA的m RNA和蛋白表達(dá)水平;(3)根據(jù)生物信息學(xué)預(yù)測(cè)結(jié)果構(gòu)建全長(zhǎng)及截短visfatin啟動(dòng)子雙熒光素酶報(bào)告基因載體:(1)將全長(zhǎng)熒光素酶載體轉(zhuǎn)染至高糖誘導(dǎo)系膜細(xì)胞,給予不同濃度的GW4064(0.5μM,1μM,5μM)或溶劑DMSO處理,24小時(shí)后檢測(cè)visfatin啟動(dòng)子活性;(2)將FXR表達(dá)質(zhì)粒和截短visfatin啟動(dòng)子雙熒光素酶報(bào)告基因載體共轉(zhuǎn)染至293細(xì)胞,給予DMSO或GW4064(5μM)處理,24小時(shí)后檢測(cè)visfatin啟動(dòng)子活性。3.動(dòng)物實(shí)驗(yàn)部分:采用db/db小鼠作為糖尿病腎病動(dòng)物模型,db/m小鼠作為對(duì)照,將db/db小鼠分為db/db(12w)組、db/db(16w)組、db/db(20w)組及db/db(20w)+GW4064治療組:(1)檢測(cè)各組小鼠血糖、體重、24小時(shí)尿白蛋白、Scr、BUN等生化指標(biāo);(2)取各組小鼠腎臟組織行PAS、Masson染色,進(jìn)行visfatin、TGF-β1、α-SMA及FN免疫組化染色;(3)Western blot檢測(cè)各組小鼠腎臟組織visfatin、NF-κB、IκBα、TGF-β1、smad2/3、p-smad2/3、α-SMA、Collagen IV及FN的蛋白表達(dá)情況。結(jié)果:1.臨床研究部分:(1)免疫組化染色結(jié)果顯示visfatin在DN患者腎臟組織表達(dá),主要定位于腎小球,腎小管間質(zhì)少量表達(dá),且visfatin表達(dá)強(qiáng)度隨著DN的分期增加表達(dá)增強(qiáng)(P0.01,與對(duì)照組比較)。(2)在早、中期DN組,visfatin表達(dá)與患者24小時(shí)尿白蛋白、Scr、BUN水平呈正相關(guān)(R=0.868,P0.01;R=0.913,P0.01;R=0.938,P0.01),與患者e GFR呈負(fù)相關(guān)(R=-0.979,P0.01);在晚期DN組,visfatin表達(dá)與患者24小時(shí)尿蛋白呈負(fù)相關(guān)(R=-0.499,P0.05),與Scr、BUN及e GFR水平無(wú)相關(guān)性(R=-0.099,P0.05;R=0.281,P0.05;R=0.026,P0.05)。2.細(xì)胞實(shí)驗(yàn)部分:(1)激活FXR通過(guò)調(diào)控visfatin表達(dá)可以抑制高糖誘導(dǎo)系膜細(xì)胞增殖及增殖相關(guān)基因、炎癥因子、促纖維化基因的表達(dá):與對(duì)照組比,HG組細(xì)胞增殖顯著增快(P0.01),GW4064組較HG組增殖速度顯著下降(P0.01),GW4064組PCNA蛋白表達(dá)水平較HG組顯著下降(P0.01);與GW4064組比,GW4064+visfatin組細(xì)胞增殖、PCNA表達(dá)顯著回升(P0.01);(2)與對(duì)照組比,HG組p-P65蛋白表達(dá)、細(xì)胞上清MCP-1水平明顯升高(P0.01),而IκBα蛋白表達(dá)顯著下降(P0.01);與HG組比,GW4064組p-P65和MCP-1表達(dá)水平顯著下調(diào)(P0.01),IκBα表達(dá)明顯回升(P0.01);與GW4064組比,visfatin組p-P65蛋白表達(dá)、上清MCP-1水平顯著上調(diào)(P0.01),而IκBα表達(dá)明顯下降(P0.01);(3)與對(duì)照組比,HG組TGF-β1、p-smad2/3、α-SMA、Collagen IV及FN的m RNA和蛋白表達(dá)水平明顯升高(P0.01),GW4064組較HG組的TGF-β1、p-smad2/3、α-SMA、Collagen IV及FN的m RNA和蛋白表達(dá)水平顯著下降(P0.01);與GW4064組比,GW4064+visfatin組TGF-β1、p-smad2/3、α-SMA、Collagen IV及FN的m RNA和蛋白表達(dá)水平明顯回升(P0.01);(4)雙熒光素酶實(shí)驗(yàn)結(jié)果顯示,與對(duì)照比,HG組visfatin啟動(dòng)子活性顯著增強(qiáng),給予FXR激動(dòng)劑GW4064可以抑制visfatin啟動(dòng)子活性,并呈濃度依賴(lài)性,其中GW4064(1μM)、GW4064(5μM)與HG組比具有統(tǒng)計(jì)學(xué)意義(P0.01);截短實(shí)驗(yàn)結(jié)果顯示visfatin啟動(dòng)子活性在1607bp-1192bp之間明顯增強(qiáng)(P0.01),提示FXR與visfatin啟動(dòng)子能的結(jié)合位點(diǎn)為visfatin啟動(dòng)子1607bp-1192bp區(qū)域。3.動(dòng)物實(shí)驗(yàn)部分:(1)與db/m組比,db/db組小鼠24小時(shí)尿白蛋白、Scr、BUN顯著升高(P0.01),且隨著周齡增加而逐漸升高;GW4064治療組小鼠24小時(shí)尿白蛋白、Scr、BUN較db/db組顯著下降(與16w及20w比,P0.01);(2)PAS及Masson染色顯示db/db小鼠腎小球明顯增大,球囊擴(kuò)展,糖原沉積,系膜及基質(zhì)增生,腎小球細(xì)胞外基質(zhì)增多,而GW4064治療組腎小球的上述改變明顯減輕;免疫組化染色顯示,visfatin主要表達(dá)于腎小球,腎小管間質(zhì)有少量表達(dá),db/db組小鼠visfatin表達(dá)隨著小鼠周齡增加逐漸增強(qiáng),GW4064治療組表達(dá)明顯下降,TGF-β1、α-SMA和FN的免疫組化染色結(jié)果顯示其趨勢(shì)同visfatin免疫組化染色;(3)Western blot結(jié)果顯示,與db/m組比,db/db組visfatin蛋白表達(dá)顯著升高,且隨著周齡增加而逐漸升高(P0.01),p-P65、TGF-β1、p-Smad2/3、α-SMA、Collagen IV和FN表達(dá)趨勢(shì)同visfatin表達(dá)(P0.01,與db/m組比);GW4064組visfatin蛋白表達(dá)較db/db組顯著下降(與16w及20w比,P0.01);p-P65、TGF-β1、p-Smad2/3、α-SMA、Collagen IV和FN的表達(dá)也明顯下調(diào)(與16w及20w比,P0.01),而IκBα表達(dá)趨勢(shì)與visfatin表達(dá)相反(與16w及20w比,P0.01)。結(jié)論:激活FXR通過(guò)調(diào)控visfatin基因啟動(dòng)子轉(zhuǎn)錄活性下調(diào)其表達(dá),從而抑制系膜細(xì)胞增殖、炎癥因子及促纖維化因子的表達(dá),延緩糖尿病腎病的進(jìn)展;其可能的結(jié)合位點(diǎn)位于visfatin啟動(dòng)子-1607bp至-1192bp區(qū)域。
[Abstract]:Background and objective: diabetic nephropathy (Diabetic Nephropathy DN) is an important complication of diabetes mellitus, which is the most common cause of end-stage renal disease (End-stage renal, disease, ESRD) one of the causes. The main pathological features of renal structural changes including glomerular hypertrophy, thickening of glomerular mesangial matrix, podocyte injury, leading to glomerular sclerosis and tubulointerstitial fibrosis. These changes lead to the increase of proteinuria and glomerular filtration rate decreased, and finally develop into end-stage renal disease. The pathogenesis of diabetic nephropathy is not completely clear, studies show that high blood pressure, high blood glucose, advanced glycation end products, lipid metabolism, fat accumulation, promoting growth factors, inflammatory fibrosis, oxidative stress plays a role in the progression of diabetic nephropathy. The farnesoid X receptor (Farnesoid X, receptor, FXR) belongs to the nuclear receptor Body superfamily, is an intracellular signaling protein, as a multifunctional transcription factor activation or inhibition of target gene expression of.FXR in liver, intestine, kidney and adrenal tissues had high expression. The current study shows that FXR in diabetic nephropathy plays an important role in the development, may be related to FXR gene involved in the regulation of lipid metabolism the expression, but the specific regulatory mechanism is still not clear that.Visfatin is a secreted protein recently isolated and identified the factor B cell clone (pre-B cell colony also called enhanced ehancing, factor, PBEF) in adipose tissue, liver, heart, skeletal muscle, brain, kidney and other tissues. The expression of visfatin in lipid research found metabolism, insulin resistance, cell proliferation, differentiation, apoptosis and inflammation play an important role in the process. The study found that visfatin increased significantly in serum concentration in patients with diabetes mellitus, participate in the regulation of inflammation The expression of inflammatory factor in endothelial dysfunction, insulin resistance, and atherosclerosis. In vitro studies have found that Visfatin in high glucose induced glomerular mesangial cells and renal tubular epithelial cells was significantly increased, the exogenous visfatin can promote the increase of mesangial cell glucose transporter GLUT-1 expression, promote the absorption of glucose, and stimulated profibrotic gene expression. Our previous study found that serum visfatin levels were significantly increased in patients with diabetic nephropathy, and a positive correlation with inflammatory factors, was negatively correlated with the level of renal function, suggesting that visfatin may be closely related to the process and the inflammatory state and diabetic nephropathy. In this study the in vitro and in vivo experiments to elucidate the molecular mechanism of FXR regulate the expression of visfatin and the progression of diabetic nephropathy in the process, to provide a new way of understanding the pathogenesis of diabetic nephropathy. Methods: 1. clinical research: from June 2014 -2015 year in December in our hospital and the clinical data of renal biopsy specimens of patients with type 2 diabetic nephropathy renal biopsy, and the pathological stages were divided into DN group of 13 cases of early stage, 16 cases in DN group, DN group of 18 cases collected late; the Department of Urology in our hospital during the period 10 cases of patients with renal tumors from tumor normal tissues as the control group; the collection of renal tissue specimens of PAS and visfatin by immunohistochemical staining; the clinical data were collected including age, gender, 24 hours urinary albumin, serum creatinine (Scr), blood urea nitrogen (BUN), and glomerular filtration rate calculation according to the Cockcroft-Grault formula (E GFR). And the statistical analysis of results are as follows: (1) comparison comparison of 24 hours urinary albumin, Scr, BUN, e, GFR and visfatin expression of semi quantitative results; (2) the DN group of patients 24 hours urine protein, Scr, BUN, e, G The results of FR, the expression of visfatin and semi quantitative analyzed the correlation between.2. cell experiment: (1) the FXR agonist GW4064, FXR antagonist Guggulsterone HMC in human mesangial cells, the expression changes of M protein and RNA Real-time PCR and Western blot detection of visfatin; (2) were treated with high glucose (HG) induced by HMC transfection of Si RNA silencing visfatin, GW4064 or exogenous visfatin treatment: (1) the expression of M RNA Real-time PCR visfatin Western blot detection, visfatin detection, NF- kappa B, the expression level of I kappa B alpha protein, detection of supernatant MCP-1 concentration by ELISA; (2) the detection of TGF- Real-time PCR 1 beta, alpha -SMA. Collagen IV and FN m RNA expression, Western blot detection of TGF- beta 1, smad2/3, p-smad2/3, alpha -SMA, the expression level of Collagen IV and FN protein; (3) detection of mesangial cell proliferation by CCK-8 m RNA, Real-time PCR and Western blot to detect the proliferation related gene PCNA And the protein expression level; (3) according to the bioinformatics prediction results of full-length and truncated visfatin promoter luciferase reporter gene vector: (1) the full-length luciferase vector was transfected into glucose induced mesangial cells treated with different concentrations of GW4064 (0.5 M, 1 M, 5 M) or solvent DMSO visfatin, detection of promoter activity after 24 hours; (2) the FXR expression plasmid and truncated visfatin promoter luciferase reporter vector was transfected into 293 cells treated with DMSO or GW4064 (5 M) treatment, detection of visfatin promoter activity in.3. animal experiment after 24 hours: using db/db mice as diabetic nephropathy the animal model of db/m mice as control group, db/db mice were divided into db/db group (12W), db/db (16W) group, db/db (20W) group and db/db (20W) +GW4064 treatment group: (1) the blood glucose, body weight of mice were detected, 24 hours urinary albumin, Scr, BUN and other biochemical indicators; (2 take each) 緇勫皬榧犺偩鑴忕粍緇囪PAS,Masson鏌撹壊,榪涜visfatin,TGF-尾1,偽-SMA鍙?qiáng)FN鍏嶇柅緇勫寲鏌撹壊;(3)Western blot媯，

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