Klotho下调Egr-1抑制糖尿病肾脏病肾小管上皮细胞转分化的作用及机制研究

发布时间：2018-01-11 21:02

本文关键词：Klotho下调Egr-1抑制糖尿病肾脏病肾小管上皮细胞转分化的作用及机制研究　出处：《南方医科大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景糖尿病肾脏病(diabetic kidney disease,DKD)已成为终末期肾病(end-stage renal disease,ESRD)的首要原因。既往认为 DKD 早期主要病变部位在肾小球,而肾小管间质纤维化是DKD发展到后期的病理学特征。然而,近年来的研究发现肾小管间质损害程度成为反映肾功能下降严重程度和判断预后最重要的指标,而肾小管上皮细胞转分化(epithelial-to-mesenchymal transition,EMT)则是肾小管间质纤维化的关键环节。Klotho是一种抗衰老因子,早期DKD患者血清Klotho水平已出现明显的下降,是DKD较早出现的生物标志物之一。Klotho可通过抑制TGF-β1/Smad3、ERK1/2信号通路,延缓DKD进展。早期生长反应蛋白-l(Early growth response protein 1,Egr-1),是一种具有锌指结构的转录因子,可通过与TGF-β1、ERK1/2形成正反馈环路促进肾脏纤维化。并且,已有研究表明,TGF-β1可通过激活ERK1/2信号通路促进肾脏纤维化。目前关于Klotho与Egr-1在DKD发病中是否有相互作用及作用机制的研究未见报道。本研究旨在探索Klotho和Egr-1在DKD肾小管上皮细胞转分化(epithelial-to-mesenchymal transition,EMT)中发挥的作用,阐明在 DKD 进展中Klotho是否通过抑制TGF-β1/Smad3-ERK1/2信号下调Egr-1发挥抗肾脏纤维化作用,为深入理解DKD发病的分子机制和寻找新的治疗靶点提供理论基础。方法1动物实验用HFD/STZ诱导C57BL/6J小鼠(n=12)制造糖尿病模型,同系正常小鼠(n=12)作为对照。成模后分别于6w、12w时留取血、尿行生化指标检测,处死后取肾脏组织冻存于-80℃冰箱,或用4%多聚甲醛固定。PAS、Masson染色法观察肾组织的形态结构变化,免疫组化法分析肾皮质中Klotho和Egr-1表达的变化。2细胞培养人肾小管上皮细胞株(HK2)购买于上海中国科学院细胞库,于含1.0g/L糖的DMEM培养基+10%FBS中培养,在37℃、5%CO2培养箱中孵育。每2-3天传代一次。3细胞转染转染前一天,细胞接种于12孔板中,转染时要求细胞密度达到60%或80%。使用lipo3000进行瞬时转染siRNA或质粒,siRNA转染浓度为50nM,质粒转染浓度为1ug/ml。4 实时荧光定量PCR(RT-qPCR)Trizol法提取组织及细胞总RNA。M-MLV逆转录酶试剂将RNA逆转录为cDNA。SYBR法进行实时荧光定量PCR反应,β-actin为内参,目的基因表达量按2-△△ct方法计算得出。5 Western BlotRIPA法提取组织及细胞总蛋白。配制10%的SDS-PAGE胶,每孔上样量为20ug。电泳分离样品蛋白,湿转到PVDF膜上。用5%脱脂奶粉或5%BSA封闭PVDF膜,一抗孵育PVDF膜,4℃摇床过夜。荧光二抗孵lh,TBST洗涤。在Odyssey近红外成像系统发光显影,Gel-Pro analyzer分析结果。6统计分析各指标数据采用均数±标准差(X±SD)表示,多组数据比较采用单因素方差分析,正态分布两样本比较采用两独立样本t检验,P0.05时被认为差异具有统计学意义。结果1 Klotho与Egr-1在HFD/STZ诱导的DM小鼠肾皮质中表达水平的变化研究(1)与对照组相比,DM组小鼠血清Klotho水平6w时已明显降低,12w进一降低,24h尿微量白蛋白水平6w时已明显升高,12w时进一步升高。(2)与对照组相比,DM组小鼠肾皮质中Klotho表达水平6w时已明显降低,12w时进一步降低,Egr-1表达水平到12w时才出现明显升高。2 Klotho在DKD进展中对肾小管EMT的作用研究(1)HK2细胞中Klotho的表达水平在HG、TGF-β1刺激后12h出现明显下降,24h进一步降低。(2)与 pcDNA-Vector 相比,瞬时转染 pcDNA-Klotho 可抑制 HG、TGF-β1诱导的HK2细胞中E-cadherin表达降低、α-SMA及FN表达升高。(3)与si-Negative相比,瞬时转染si-Klotho可使HG、TGF-[β1诱导的HK2细胞中E-cadherin表达进一步降低、α-SMA及FN表达进一步升高。3 Egr-1在DKD进展中对肾小管EMT的作用研究(1)HK2细胞中Egr-1的表达在HG、TGF-β1刺激后0.5h出现明显的升高,6h回落到基线水平。(2)与si-Negative相比,瞬时转染si-Egr-1可减弱HG、TGF-β1诱导的HK2细胞中E-cadherin表达降低、α-SMA及FN表达升高。(3)与 pENTER-Vector 相比,瞬时转染 pENTER-Egr-1 可使 HG、TGF-βl诱导的HK2细胞中E-cadherin表达进一步降低、α-SMA及FN表达进一步升高。4 Klotho在DKD进展中下调Egr-1的机制研究(1)与 pcDNA-Vector 相比,瞬时转染 pcDNA-Klotho 可抑制 HG、TGF-βl 诱导的 HK2 细胞中 Egr-1 表达升高、p-Smad3/Smad3 及(p-ERKl/2)/(ERK1/2)蛋白比值升高。(2)与si-Negative相比,瞬时转染si-Klotho可使HG、TGF-βl诱导的HK2 细胞中 Egr-1 表达进一步升高,p-Smad3/Smad3 及(p-ERKl/2)/(ERKl/2)蛋白比值进一步升高,si-Klotho的上述作用可被ERK1/2抑制剂PD98059明显减弱。结论l早期DKD小鼠肾皮质中Klotho表达明显减少,Egr-1表达明显增加,且Klotho表达水平的降低早期Egr-1表达水平的升高。2 HG、TGF-βl对HK2细胞中Klotho表达的抑制作用呈时间依赖性,Klotoh在DKD进展中对肾小管EMT具有抑制作用。3 HG、TGF-β1可诱导HK2细胞中Egr-1瞬时表达,Egr-1在DKD进展中对肾小管EMT具有促进作用。4 Klotho 通过抑制 TGF-β1/Smad3-ERKl/2 信号通路下调 HG、TGF-βl 诱导的HK2中Egr-1的表达,这可能是Klotho在DKD进展中抗肾脏纤维化的重要机制之一。
[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.

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.2;R692.9

【相似文献】