Klotho基因超甲基化在硫酸吲哚酚诱导的血管钙化中的作用机制研究

发布时间：2018-03-07 16:47

本文选题：终末期肾病　切入点：血管钙化　出处：《复旦大学》2014年博士论文　论文类型：学位论文

【摘要】：第一部分尿毒症患者血管局部Klotho基因超甲基化与血管钙化的关系背景：最新研究证实人类血管平滑肌细胞是Klotho直接作用的靶点,血管局部Klotho表达下调参与血管钙化的发生发展,但血管局部Klotho低表达的原因尚不清楚。DNA甲基化是调节基因表达的一种表观遗传方式,启动子区CpG岛(即300～3000 bp富含CpG二核苷酸的区域的甲基化有沉默基因表达的作用。本研究旨在以ESRD患者为研究对象,探讨ESRD患者桡动脉局部Klotho基因超甲基化与血管钙化的关系。方法：2013.1～2013.12在复旦大学附属中山医院肾内科病房住院治疗并行动静脉内瘘手术的30例透析前ESRD患者为尿毒症组,同期于我院心外科病房行冠状动脉搭桥手术的10例年龄、性别匹配的患者作为对照组,均取得患者知情同意。术中分别取桡动脉和同级别的内乳动脉行H.E.染色观察血管内中膜厚度和血管管壁厚度/血管直径,茜素红钙盐沉积染色评估血管钙化,免疫组织化学染色评价a-SMA、OPN、Cbfα1、Klotho和DNMT1蛋白的表达水平。同时利用焦磷酸测序方法检测血管局部Klotho基因甲基化率和超高效液相色谱检测技术检测尿毒症组患者血清IS浓度。结果：尿毒症组患者内中膜厚度较对照组显著增厚(0.68±053mm vs.0.18±0.13mm,P0.01),血管管壁厚度/血管直径亦显著高于对照组(0.34±0.19 vs.0.23±0.09,P0.01)。尿毒症组中21例(70%)茜素红染色阳性,均分布在血管中膜,对照组无一例发生血管钙化(P0.01)。血管局部a-SMA、OPN和Cbfα1染色均主要分布于血管中膜,尿毒症组αα-SMA染色阳性率显著低于对照组(30% vs.100%,P0.01),OPN和Cbfα1染色阳性率显著高于对照组(87% vs.0%,P0.01)(93% vs.0%,P0.01)。尿毒症组患者血管局部Klotho基因甲基化率显著高于对照组(43.2±1.7%,8.7±0.8%,P0.001)。血管局部Klotho、 DNMT1染色均主要分布于血管中膜,尿毒症组Klotho染色阳性率显著低于对照组(33.3% vs.100%,P0.01),DNMT1染色阳性率显著高于对照组(70% vs.0%,P0.01)。其中21例茜素红染色阳性的ESRD患者者血管局部Klotho甲基化率和血清IS浓度显著高于9例染色阴性者(41.8±3.7% vs.29.4±1.5%,P0.001) (34.6±4.13 pg/m,29.8±4.23 pg/mlP0.001)。进一步Pearson单因素相关分析显示尿毒症组患者血清IS浓度与血管局部Klotho甲基化率呈正相关(r=0.587,P0.01)。结论：尿毒症患者普遍存在血管钙化和血管局部Klotho基因超甲基化,血管局部Klotho基因超甲基化与血管钙化密切相关。第二部分Klotho基因超甲基化在硫酸吲哚酚诱导的血管平滑肌细胞成骨转化中的作用机制背景：IS与ESRD患者心血管疾病死亡率和总死亡率相关,可通过诱导血管平滑肌细胞成骨细胞转分化而促进血管钙化,但是IS诱导血管钙化的具体机制目前尚不清楚。第一部分的研究证明尿毒症患者普遍存在血管钙化和血管局部Klotho基因超甲基化,血清IS浓度与血管局部Klotho基因甲基化率密切相关。本研究旨在以人主动脉血管平滑肌细胞为研究对象,从细胞层面探讨Klotho基因超甲基化在IS诱导的血管平滑肌细胞成骨转化中的作用和相关机制。方法：体外培养人主动脉血管平滑肌细胞,IS按0、200、500和1000μM的浓度梯度干预6天,采用茜素红染色观察钙盐沉积,Real-time PCR和Western blot检测a-SMA、OPN、Cbfα1ΟKlotho和不同DNMT的mRNA和蛋白表达水平,同时利用焦磷酸测序方法检测HASMC Klotho基因甲基化率。IS1000μM组培养基中加入不同浓度梯度的5-氮杂脱氧胞苷进行干预,观察茜素红染色、各基因表达水平和Klotho基因甲基化率的改变。结果：IS 1000μM组细胞外基质呈橙色,细胞结节处浓染,为钙盐沉积染色阳性。IS可以下调HASMC a-SMA mRNA和蛋白表达水平,上调OPN和Cbfa1的mRNA和蛋白表达水平,使HASMC逐渐丧失平滑肌细胞表型而发生成骨转化。IS 200、500和1000μM刺激6天可显著升高HASMC Klotho基因甲基化率(9±1%vs.4±0.7%, P0.01)、(18±1.3 vs.4±0.7%, P0.01)、(41±2%vs.4±0.7%, P0.001)。IS 500和1000gM可显著下调HASMC Klotho mRNA和蛋白表达水平。同时IS可显著上调HASMC DNMT1 mRNA和蛋白表达水平。5-氮杂脱氧胞苷10μM干预可减轻IS诱导的细胞外钙盐沉积,上调a-SMA蛋白表达水平并下调Cbfa1蛋白表达水平,同时减低HASMC Klotho基因甲基化率(20+1%vs.41±2%,P0.01)、上调HASMC Klotho蛋白表达水平,提示HASMC Klotho超甲基化可减轻硫酸吲哚酚诱导的血管平滑肌细胞成骨转化。结论：硫酸吲哚酚可诱导血管平滑肌细胞成骨转化,并可通过上调血管平滑肌细胞DNMT11的活性诱导Kloth o超甲基化,下调Klotho蛋白表达水平,阻断Klotho超甲基化可减轻硫酸吲哚酚诱导的血管平滑肌细胞成骨转化。第三部分Klotho基因超甲基化在硫酸吲哚酚诱导的尿毒症大鼠血管钙化中的作用机制背景：尿毒症患者普遍存在血管钙化和血管局部Klotho基因超甲基化,血清IS浓度与血管局部Klotho基因甲基化率密切相关。硫酸吲哚酚可诱导血管平滑肌细胞成骨转化,并可通过上调血管平滑肌细胞DNMT1的活性诱导Klotho超甲基化,下调Klotho蛋白表达水平,阻断Klotho超甲基化可减轻硫酸吲哚酚诱导的血管平滑肌细胞成骨转化。本研究旨在以尿毒症大鼠为研究对象,从体内层面探讨Klotho基因超甲基化在IS诱导的尿毒症大鼠血管钙化中的作用和相关机制。方法：采用硫酸吲哚酚联合5/6肾切除的方法构建尿毒症大鼠血管钙化模型,24只SD大鼠采用两步法行5/6肾切除术,随机分为对照组、IS组和IS+5Aza-2dc组。24周后留取血液和尿液标本,大鼠处死后留取胸主动脉标本。行H.E.染色观察血管内中膜厚度和血管管壁厚度/血管直径,茜素红钙盐沉积染色评估血管钙化,免疫组织化学染色、Real-time PCR和Western blot评价a-SMA、 OPN、 Cbfα1、Klotho和DNMT1基因mRNA和蛋白的表达水平。同时利用焦磷酸测序方法检测胸主动脉Klotho基因甲基化率和超高效液相色谱检测技术检测血清IS浓度。结果：IS组胸主动脉IMT和血管管壁厚度/血管直径显著高于对照组(0.21±0.03mm vs.0.14±0.02mm, P0.05) (0.34±0.19 vs.0.22±0.07, P0.05), 对照组2例(25%)茜素红钙盐沉积染色阳性,IS组8例(100%)阳性,IS组阳性率显著高于对照组(P0.01)。IS组胸主动脉a-SMA蛋白表达水平较对照组显著下调,IS组胸主动脉Cbfα1蛋白表达水平较对照组显著上调。IS组胸主动脉Klotho甲基化率较对照组显著升高(29±5% vs.6±2%,P0.001),IS组胸主动脉Klotho蛋白表达水平较对照组显著下调,DNMT1蛋白表达水平较对照组显著上调。5-氮杂脱氧胞苷干预后,IS+5-Aza-2dc组胸主动脉IMT和血管管壁厚度/血管直径显著低于IS组(0.21±0.03 mm vs.0.15±0.05mm, P0.05) (0.34±0.19 vs 0.23±0.09, P0.05),茜素红钙盐沉积染色5例(62.5%)阳性,显著低于IS组(P0.01)。IS+5Aza-2dc组胸主动脉a-SMA蛋白表达水平较IS组显著上调,Cbfα1蛋白表达水平较IS组显著下调,与对照组无差异。IS+5Aza-2dc组胸主动脉Klotho甲基化率较IS组显著降低(11±2% vs.29±5%,P0.01),与对照组无显著差异,且Klotho蛋白表达水平较IS组显著上调,与对照组无差异。结论：硫酸吲哚酚可诱导尿毒症大鼠血管钙化,并可通过上调血管局部DNMT11的活性诱导Klotho超甲基化,下调Klotho蛋白表达水平,阻断Klotho超甲基化可减轻硫酸吲哚酚诱导的尿毒症大鼠血管钙化。
[Abstract]:The first part background uremic vessel local hypermethylation of Klotho gene and vascular calcification: new research confirmed that human vascular smooth muscle cells are targets of direct action of Klotho, Klotho expression of local vessels involved in the occurrence and development of vascular calcification, but the cause of local vascular Klotho low expression is unclear.DNA methylation in the regulation of gene expression an epigenetic, promoter region CpG island methylation (i.e. 300~3000 BP CpG rich regions are dinucleotide silencing gene expression. This study aims to ESRD patients as the research object, to explore the relationship between the radial artery in patients with ESRD. The local hypermethylation of Klotho gene and vascular calcification method: 2013.1 ~ 2013.12 in the Department of Nephrology Zhongshan Hospital Affiliated to Fudan University ward hospitalization and surgery for arteriovenous fistula in 30 cases of ESRD patients before hemodialysis for uremia group, compared to me 10 cases of hospital cardiac surgery ward underwent coronary artery bypass surgery, sex matched patients as control group, were respectively to obtain informed consent of patients. The same level of radial artery and internal mammary artery were performed in H.E. staining to observe the intima-media thickness and vascular wall thickness / vascular diameter, the deposition of calcium salt staining Alizarin red the assessment of vascular calcification, immunohistochemical evaluation of a-SMA, OPN, Cbf alpha 1, the expression level of Klotho and DNMT1 protein. At the same time using the pyrosequencing method to detect the Klotho gene methylation rate of local vascular examination and detection technology of ultra high performance liquid chromatography to measure the serum concentration of IS in patients with uremia group. Results: the intima-media thickness in patients with uremia group compared with the control group increased significantly (0.68 + 053mm vs.0.18 + 0.13MM, P0.01), vascular wall thickness / vascular diameter was significantly higher than that of the control group (0.34 + 0.19 vs.0.23 + 0.09, P0.01). In 21 cases of uremia group (70%). Pigment red staining, are distributed in the intima, the control group with no occurrence of vascular calcification (P0.01). The local a-SMA vessels, OPN and Cbf alpha 1 staining were mainly distributed in the intima, the positive rate of uremia group alpha alpha -SMA staining was significantly lower than the control group (30% vs.100%, P0.01), the positive rate of OPN alpha 1 and Cbf staining was significantly higher than the control group (87% vs.0%, P0.01) (93% vs.0%, P0.01). Patients with uremia group vascular Klotho gene methylation rate was significantly higher than that of the control group (43.2 + 1.7%, 8.7 + 0.8%, P0.001). The local Klotho vessels, DNMT1 staining was mainly distributed in the intima, positive rate the uremia group Klotho staining was significantly lower than the control group (33.3% vs.100%, P0.01), the positive rate of DNMT1 was significantly higher than that of the control group (70% vs.0%, P0.01). 21 cases of alizarin red staining was positive in ESRD patients with local vascular Klotho methylation rate and serum IS concentration was significantly higher than that of negative staining in 9 cases (41.8. 3.7% vs.29.4 + 1.5%), P0.001 (34.6 + 4.13 + 4.23 pg/m, 29.8 pg/mlP0.001). Further Pearson single factor correlation analysis showed that serum IS concentration in patients with uremia and vascular local Klotho methylation rate was positively correlated (r=0.587, P0.01). Conclusion: uremic patients with widespread vascular calcification and vascular local hypermethylation of Klotho gene Klotho, the local vascular gene is closely related with methyl vascular calcification. Part second of the Klotho gene hypermethylation in vascular smooth muscle cells induced by indoxyl sulfate into the background in the transformation mechanism of bone: IS and ESRD in patients with cardiovascular disease and total mortality, bone cell transdifferentiation and promote vascular calcification induced by vascular smooth muscle cells, but the specific mechanism of IS induced vascular calcification is unclear. The first part of the study that is prevalent in patients with vascular calcification and uremia Local vascular Klotho hypermethylation, serum concentration of IS and vascular Klotho gene methylation rate was closely related. The purpose of this research is to human aortic vascular smooth muscle cells as the research object, to explore the hypermethylation of Klotho gene in osteoblast conversion role and related mechanisms in vascular smooth muscle cells induced by IS from the cell level. Methods: aortic vascular smooth muscle cells cultured in vitro by IS 0200500 concentration gradient and 1000 M intervention for 6 days, using alizarin red staining calcium salt deposition, Real-time PCR and Western blot detection of a-SMA, OPN, Cbf, Klotho and alpha 1 April mRNA and protein expression level of DNMT, while using the pyrosequencing method for detecting HASMC Klotho the methylation rate of.IS1000 gene M were cultured with different concentration gradient in 5- - deoxycitydine intervention, observed by alizarin red staining, the level of gene expression and Klotho gene The methylation rate changes. Results: IS 1000 M group of extracellular matrix orange cell nodules stain for the deposition of calcium salt staining positive.IS can downregulate the level of HASMC a-SMA mRNA and protein expression level, protein expression level of mRNA and upregulation of OPN and Cbfa1, the HASMC gradually lose the phenotype of smooth muscle cell and osteoblast conversion of.IS 200500 and 1000 M stimulation for 6 days can significantly increase the HASMC Klotho gene methylation rate (9 + 1%vs.4 + 0.7%, P0.01), (18 + 1.3 vs.4 + 0.7%, P0.01), (41 + 2%vs.4 + 0.7%, P0.001).IS 500 and 1000gM can significantly lower HASMC Klotho mRNA and protein expression level. At the same time, IS significantly increased HASMC DNMT1 mRNA and protein expression level of.5- aza deoxycytidine 10 M intervention can reduce the extracellular calcium salt deposition induced by IS, expression of a-SMA and down-regulation of Cbfa1 protein expression level of HASMC, while reducing the Klotho gene methylation rate (20+1%v S.41 + 2%, P0.01), HASMC up-regulated the expression level of Klotho protein, suggesting that HASMC Klotho hypermethylation can relieve the vascular smooth muscle cells of indoxyl sulfate induced osteoblast conversion. Conclusion: indoxyl sulfate can induce osteogenic transformation of vascular smooth muscle cells, and vascular smooth muscle cells through upregulation of DNMT11 activity induced by Kloth o methyl and reduce the expression of Klotho protein, blocking Klotho hypermethylation can relieve the vascular smooth muscle cells of indoxyl sulfate induced osteoblast conversion. Part third of the Klotho gene hypermethylation background mechanism of vascular calcification in uremic rats induced by indoxyl sulfate in uremic patients: vascular calcification is a common vascular and local Klotho gene hypermethylation, the concentration of serum IS and vascular local methylation rate of Klotho gene is closely related. Indoxyl sulfate can be transformed into bone induced vascular smooth muscle cells, and can pass DNMT1 up-regulated the activity of vascular smooth muscle cells induced by Klotho hypermethylation, down regulated expression of Klotho protein, blocking Klotho hypermethylation can relieve the vascular smooth muscle cells of indoxyl sulfate induced osteoblast conversion. The purpose of this study was to uremic rats as the research object, to explore the hypermethylation of Klotho gene in vascular calcification in uremic rats induced by IS the role and mechanism of in vivo level. Methods: the vascular calcification in uremic rats model by using the method of indoxyl sulfate combined with 5/6 nephrectomy, 24 SD rats using two step method for 5/6 nephrectomy, were randomly divided into control group, IS group and IS+5Aza-2dc group after.24 weeks of blood and urine samples. The rats were sacrificed for thoracic aortic specimens. H.E. staining was used to observe the intima-media thickness and vascular wall thickness / vascular diameter, red calcium salt deposition alizarin staining was used to assess vascular calcification, immune group PCR and Real-time histochemical staining, Western blot OPN, Cbf a-SMA, alpha 1, the expression level of Klotho and DNMT1 gene mRNA and protein. At the same time using the pyrosequencing method for detection of thoracic aortic Klotho gene methylation rate and serum IS ultra high performance liquid chromatography detection concentration. Results: IS group of thoracic aorta IMT and vascular wall thickness / vascular diameter was significantly higher than the control group (0.21 + 0.03mm vs.0.14 + 0.02mm, P0.05) (0.34 + 0.19 vs.0.22 + 0.07, P0.05), the control group of 2 cases (25%) red calcium salt deposition alizarin staining, IS group of 8 cases (100%) positive, the positive rate of IS group was significantly higher than that of the control group (P0.01) group the expression of.IS a-SMA protein in thoracic aorta significantly lower levels than the control group, IS group of thoracic aortic Cbf alpha 1 expression raised significantly compared to the control group.IS group aortic Klotho methylation rate was significantly higher than that of control group (29 + 5% vs.6 + 2%, P0.001), IS group of chest The expression level of Klotho protein in the aorta than in the control group significantly decreased, the expression level of DNMT1 protein was significantly up-regulated.5- - deoxycitydine intervention group IS+5-Aza-2dc thoracic aortic IMT and vascular wall thickness / vascular diameter was significantly lower than that of IS group (0.21 + 0.03 mm vs.0.15 + 0.05mm, P0.05) (0.34 + 0.19 vs 0.23 + 0.09 P0.05, red), the deposition of calcium alizarin staining in 5 cases (62.5%) were significantly lower than those in group IS (P0.01).IS+5Aza-2dc expression level of a-SMA protein in thoracic aorta group than in IS group significantly increased, Cbf protein expression level of alpha 1 decreased significantly compared with IS group,.IS+5Aza-2dc group and control group had no difference between thoracic aortic Klotho methylation rate is the IS group was significantly lower (11 + 2% vs.29 + 5%, P0.01), and the control group had no significant difference, and the expression level of Klotho protein increased significantly compared with IS group, no difference with the control group. Conclusion: indoxyl sulfate can induce vascular calcification in uremic rats, and It can induce Klotho hypermethylation and down regulate the expression level of Klotho protein by up regulating the activity of local DNMT11, and blocking Klotho hypermethylation can reduce the vascular calcification of indolol induced uremic rats.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692.5

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