發(fā)布時間：2018-01-11 21:02

  本文關(guān)鍵詞：Klotho下調(diào)Egr-1抑制糖尿病腎臟病腎小管上皮細(xì)胞轉(zhuǎn)分化的作用及機制研究　出處：《南方醫(yī)科大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： Klotho Egr-1 糖尿病腎臟病 腎小管上皮細(xì)胞轉(zhuǎn)分化 腎臟纖維化

【摘要】：研究背景糖尿病腎臟病(diabetic kidney disease,DKD)已成為終末期腎病(end-stage renal disease,ESRD)的首要原因。既往認(rèn)為 DKD 早期主要病變部位在腎小球,而腎小管間質(zhì)纖維化是DKD發(fā)展到后期的病理學(xué)特征。然而,近年來的研究發(fā)現(xiàn)腎小管間質(zhì)損害程度成為反映腎功能下降嚴(yán)重程度和判斷預(yù)后最重要的指標(biāo),而腎小管上皮細(xì)胞轉(zhuǎn)分化(epithelial-to-mesenchymal transition,EMT)則是腎小管間質(zhì)纖維化的關(guān)鍵環(huán)節(jié)。Klotho是一種抗衰老因子,早期DKD患者血清Klotho水平已出現(xiàn)明顯的下降,是DKD較早出現(xiàn)的生物標(biāo)志物之一。Klotho可通過抑制TGF-β1/Smad3、ERK1/2信號通路,延緩DKD進展。早期生長反應(yīng)蛋白-l(Early growth response protein 1,Egr-1),是一種具有鋅指結(jié)構(gòu)的轉(zhuǎn)錄因子,可通過與TGF-β1、ERK1/2形成正反饋環(huán)路促進腎臟纖維化。并且,已有研究表明,TGF-β1可通過激活ERK1/2信號通路促進腎臟纖維化。目前關(guān)于Klotho與Egr-1在DKD發(fā)病中是否有相互作用及作用機制的研究未見報道。本研究旨在探索Klotho和Egr-1在DKD腎小管上皮細(xì)胞轉(zhuǎn)分化(epithelial-to-mesenchymal transition,EMT)中發(fā)揮的作用,闡明在 DKD 進展中Klotho是否通過抑制TGF-β1/Smad3-ERK1/2信號下調(diào)Egr-1發(fā)揮抗腎臟纖維化作用,為深入理解DKD發(fā)病的分子機制和尋找新的治療靶點提供理論基礎(chǔ)。方法1動物實驗用HFD/STZ誘導(dǎo)C57BL/6J小鼠(n=12)制造糖尿病模型,同系正常小鼠(n=12)作為對照。成模后分別于6w、12w時留取血、尿行生化指標(biāo)檢測,處死后取腎臟組織凍存于-80℃冰箱,或用4%多聚甲醛固定。PAS、Masson染色法觀察腎組織的形態(tài)結(jié)構(gòu)變化,免疫組化法分析腎皮質(zhì)中Klotho和Egr-1表達的變化。2細(xì)胞培養(yǎng)人腎小管上皮細(xì)胞株(HK2)購買于上海中國科學(xué)院細(xì)胞庫,于含1.0g/L糖的DMEM培養(yǎng)基+10%FBS中培養(yǎng),在37℃、5%CO2培養(yǎng)箱中孵育。每2-3天傳代一次。3細(xì)胞轉(zhuǎn)染轉(zhuǎn)染前一天,細(xì)胞接種于12孔板中,轉(zhuǎn)染時要求細(xì)胞密度達到60%或80%。使用lipo3000進行瞬時轉(zhuǎn)染siRNA或質(zhì)粒,siRNA轉(zhuǎn)染濃度為50nM,質(zhì)粒轉(zhuǎn)染濃度為1ug/ml。4 實時熒光定量PCR(RT-qPCR)Trizol法提取組織及細(xì)胞總RNA。M-MLV逆轉(zhuǎn)錄酶試劑將RNA逆轉(zhuǎn)錄為cDNA。SYBR法進行實時熒光定量PCR反應(yīng),β-actin為內(nèi)參,目的基因表達量按2-△△ct方法計算得出。5 Western BlotRIPA法提取組織及細(xì)胞總蛋白。配制10%的SDS-PAGE膠,每孔上樣量為20ug。電泳分離樣品蛋白,濕轉(zhuǎn)到PVDF膜上。用5%脫脂奶粉或5%BSA封閉PVDF膜,一抗孵育PVDF膜,4℃搖床過夜。熒光二抗孵lh,TBST洗滌。在Odyssey近紅外成像系統(tǒng)發(fā)光顯影,Gel-Pro analyzer分析結(jié)果。6統(tǒng)計分析各指標(biāo)數(shù)據(jù)采用均數(shù)±標(biāo)準(zhǔn)差(X±SD)表示,多組數(shù)據(jù)比較采用單因素方差分析,正態(tài)分布兩樣本比較采用兩獨立樣本t檢驗,P0.05時被認(rèn)為差異具有統(tǒng)計學(xué)意義。結(jié)果1 Klotho與Egr-1在HFD/STZ誘導(dǎo)的DM小鼠腎皮質(zhì)中表達水平的變化研究(1)與對照組相比,DM組小鼠血清Klotho水平6w時已明顯降低,12w進一降低,24h尿微量白蛋白水平6w時已明顯升高,12w時進一步升高。(2)與對照組相比,DM組小鼠腎皮質(zhì)中Klotho表達水平6w時已明顯降低,12w時進一步降低,Egr-1表達水平到12w時才出現(xiàn)明顯升高。2 Klotho在DKD進展中對腎小管EMT的作用研究(1)HK2細(xì)胞中Klotho的表達水平在HG、TGF-β1刺激后12h出現(xiàn)明顯下降,24h進一步降低。(2)與 pcDNA-Vector 相比,瞬時轉(zhuǎn)染 pcDNA-Klotho 可抑制 HG、TGF-β1誘導(dǎo)的HK2細(xì)胞中E-cadherin表達降低、α-SMA及FN表達升高。(3)與si-Negative相比,瞬時轉(zhuǎn)染si-Klotho可使HG、TGF-[β1誘導(dǎo)的HK2細(xì)胞中E-cadherin表達進一步降低、α-SMA及FN表達進一步升高。3 Egr-1在DKD進展中對腎小管EMT的作用研究(1)HK2細(xì)胞中Egr-1的表達在HG、TGF-β1刺激后0.5h出現(xiàn)明顯的升高,6h回落到基線水平。(2)與si-Negative相比,瞬時轉(zhuǎn)染si-Egr-1可減弱HG、TGF-β1誘導(dǎo)的HK2細(xì)胞中E-cadherin表達降低、α-SMA及FN表達升高。(3)與 pENTER-Vector 相比,瞬時轉(zhuǎn)染 pENTER-Egr-1 可使 HG、TGF-βl誘導(dǎo)的HK2細(xì)胞中E-cadherin表達進一步降低、α-SMA及FN表達進一步升高。4 Klotho在DKD進展中下調(diào)Egr-1的機制研究(1)與 pcDNA-Vector 相比,瞬時轉(zhuǎn)染 pcDNA-Klotho 可抑制 HG、TGF-βl 誘導(dǎo)的 HK2 細(xì)胞中 Egr-1 表達升高、p-Smad3/Smad3 及(p-ERKl/2)/(ERK1/2)蛋白比值升高。(2)與si-Negative相比,瞬時轉(zhuǎn)染si-Klotho可使HG、TGF-βl誘導(dǎo)的HK2 細(xì)胞中 Egr-1 表達進一步升高,p-Smad3/Smad3 及(p-ERKl/2)/(ERKl/2)蛋白比值進一步升高,si-Klotho的上述作用可被ERK1/2抑制劑PD98059明顯減弱。結(jié)論l早期DKD小鼠腎皮質(zhì)中Klotho表達明顯減少,Egr-1表達明顯增加,且Klotho表達水平的降低早期Egr-1表達水平的升高。2 HG、TGF-βl對HK2細(xì)胞中Klotho表達的抑制作用呈時間依賴性,Klotoh在DKD進展中對腎小管EMT具有抑制作用。3 HG、TGF-β1可誘導(dǎo)HK2細(xì)胞中Egr-1瞬時表達,Egr-1在DKD進展中對腎小管EMT具有促進作用。4 Klotho 通過抑制 TGF-β1/Smad3-ERKl/2 信號通路下調(diào) HG、TGF-βl 誘導(dǎo)的HK2中Egr-1的表達,這可能是Klotho在DKD進展中抗腎臟纖維化的重要機制之一。
[Abstract]:The research background of diabetic kidney disease (diabetic kidney, disease, DKD) has become the end-stage renal disease (end-stage renal, disease, ESRD) of the primary reason. Previous studies showed that DKD early lesions are mainly in glomeruli and renal tubular interstitial fibrosis is DKD to the characteristic of late pathology. However, recent studies have found that renal tubule matter damage as a reflection of the decline of renal function and the most important prognostic markers, and transdifferentiation of renal tubular epithelial cells (epithelial-to-mesenchymal, transition, EMT) is the key link of renal tubule interstitial fibrosis.Klotho is an anti-aging factor, early DKD patients serum Klotho level has been a significant decline, is a biological marker DKD earlier one of.Klotho through inhibition of TGF- beta 1/Smad3, ERK1/2 signaling pathway, delay the progression of DKD. Early growth response protein -l (Early growth respo NSE protein, 1, Egr-1) is a zinc finger transcription factor structure, with TGF- beta 1, ERK1/2 form a positive feedback loop to promote renal fibrosis. Furthermore, studies have shown that TGF- beta 1 can activate ERK1/2 signaling pathway to promote renal fibrosis. At present there is no research on whether Klotho can interact with Egr-1 and the mechanism in the pathogenesis of DKD is reported. This study aims to explore Klotho and Egr-1 transdifferentiation in renal tubular epithelial cells of DKD (epithelial-to-mesenchymal transition EMT) play a role, to clarify whether Klotho through inhibition of TGF- beta 1/Smad3-ERK1/2 signal down-regulation of Egr-1 play anti renal fibrosis in the progression of DKD, and provide a theoretical basis for further understanding the molecular the pathogenesis of DKD and to find new therapeutic targets. C57BL/6J mice induced by HFD/STZ method 1 animal experiments (n=12) manufacturing system with diabetes model, normal mice (n =12) as control. After modeling respectively in 6W, 12W blood and urine biochemical indexes, and sacrificed kidney tissue freezing at -80 deg.c, refrigerator, or fixed by 4% paraformaldehyde.PAS, morphological changes of renal tissue were observed by Masson staining, immunohistochemical analysis in.2 cells the expression of Klotho and Egr-1 in renal cortex of human renal tubular epithelial cell culture (HK2) purchased from Shanghai Academy of Sciences Chinese cell library, containing 1.0g/L sugar DMEM culture medium +10%FBS, at 37 degrees C, were incubated with 5%CO2. Passage every 2-3 days once a day after transfection of.3 cells before. Cells were seeded in 12 well plates, when the cell density reached 60% for transfection or 80%. lipo3000 transfected siRNA or siRNA plasmid transfection, the concentration of 50nM, concentration of plasmid transfection by real-time fluorescence quantitative PCR 1ug/ml.4 (RT-qPCR) on total RNA.M-MLV extracted from tissues and cells detected by Trizol Agent RNA reverse transcription cDNA.SYBR method for real-time fluorescence quantitative PCR reaction, beta -actin as standard, gene expression obtained.5 Western BlotRIPA method to extract the total protein of tissues and cells was calculated by 2- delta CT method. With 10% SDS-PAGE glue, each hole sample was isolated from the sample protein 20ug. electrophoresis, wet to PVDF film closed. PVDF film with 5% skimmed milk or 5%BSA, an anti PVDF membrane was incubated for 4 DEG C, shaking overnight. Two fluorescence antibody for LH TBST in Odyssey, wash. Near infrared imaging system of light imaging, analyzer analysis results of Gel-Pro.6 statistical analysis of the mean and standard deviation of each index data using (X + SD) said multiple sets of data, compared with single factor analysis of variance, normal distribution of two samples using two independent samples t test, P0.05 was considered statistically significant. Results of the 1 Klotho and the expression level of Egr-1 in HFD/STZ induced DM mice in renal cortex The change of (1) compared with the control group, the level of serum Klotho in DM group 6W decreased, 12W decreased in 24h, urinary albumin levels of 6W increased significantly when 12W increased further. (2) compared with the control group, the level of 6W Klotho expression in renal cortex of mice in DM group was obviously reduced 12W further decreased the expression level of Egr-1 to 12W was increased significantly.2 effect of Klotho on renal tubular EMT in the progression of DKD (1) Klotho expression in HK2 cells HG, TGF- beta 1 after stimulation of 12h decreased significantly, 24h decreased further. (2) compared with pcDNA-Vector. Transient transfection of pcDNA-Klotho can inhibit HG, TGF- beta E-cadherin 1 induced HK2 cells decreased expression of increased expression of alpha -SMA and FN. (3) compared with si-Negative, the transient transfection of si-Klotho HG, TGF-[E-cadherin beta 1 expression in HK2 cells induced by further reduced expression of alpha -SMA and FN Further increase the effect of.3 Egr-1 on renal tubular EMT in the progression of DKD (1) Egr-1 expression in HK2 cells in HG, TGF- beta 1 0.5h after stimulation significantly increased, 6h fell to baseline. (2) compared with si-Negative transfected si-Egr-1 can decrease HG, TGF- beta 1 induced E-cadherin the decreased expression of HK2 cells, increased expression of -SMA alpha and FN. (3) compared with pENTER-Vector transfected pENTER-Egr-1 can make HG, to further reduce the E-cadherin expression of TGF- beta l induced HK2 cells, mechanism of expression of alpha -SMA and FN increased.4 Klotho down-regulation of Egr-1 in development of DKD (1) compared with pcDNA-Vector pcDNA-Klotho, transient transfection inhibited HG, Egr-1 increased the expression of TGF- beta L in HK2 cells induced by p-Smad3/Smad3, and (p-ERKl/2) (ERK1/2) / protein ratio increased. (2) compared with si-Negative transfected si-Klotho can make HG, TGF- beta l induced H Further increase of Egr-1 expression in K2 cells, and p-Smad3/Smad3 (p-ERKl/2) (ERKl/2) / protein ratio increased further, the effect of si-Klotho ERK1/2 inhibitor PD98059 can be significantly reduced. Conclusion early L Klotho DKD expression in renal cortex of mice significantly reduced the expression of Egr-1 increased significantly, and the expression level of Klotho decreased early Egr-1 expression level.2 increased HG, TGF- beta inhibitory effect of L on the expression of Klotho in HK2 cells was time dependent, Klotoh has the inhibitory effect of.3 HG on renal tubular EMT in the progression of DKD, TGF- beta 1 can induce transient expression of Egr-1 in HK2 cells, Egr-1 can promote.4 Klotho through inhibition of TGF- beta 1/Smad3-ERKl/2 signaling pathway on the down-regulation of HG EMT of renal tubule in the progression of DKD, the expression of Egr-1 TGF- induced by L beta HK2, which may be an important mechanism of anti renal fibrosis Klotho in the progression of DKD.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R587.2;R692.9

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