【作者】 陈静；

【导师】 丁小强；

【作者基本信息】 复旦大学 ， 内科学， 2014， 博士

【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,P<0.01),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,P<0.01)。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(P<0.01)。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%,P<0.01),OPN和Cbfα1染色阳性率显著高于对照组(87% vs.0%,P<0.01)(93% vs.0%,P<0.01)。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%,P<0.001)。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%,P<0.01),DNMT1染色阳性率显著高于对照组(70% vs.0%,P<0.01)。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%,P<0.001) (34.6±4.13 pg/m,29.8±4.23 pg/mlP<0.001)。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,P<0.01)。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%, P<0.01)、(18±1.3 vs.4±0.7%, P<0.01)、(41±2%vs.4±0.7%, P<0.001)。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%,P<0.01)、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, P<0.05) (0.34±0.19 vs.0.22±0.07, P<0.05), 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(P<0.01)。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%,P<0.001),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, P<0.05) (0.34±0.19 vs 0.23±0.09, P<0.05),茜素红钙盐沉积染色5例(62.5%)阳性,显著低于IS组(P<0.01)。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%,P<0.01),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。更多还原

【Abstract】 Part I The relationship between Klotho methylation levels in uremic patients and vascular calcificationBackground:Recent study confirmed that Klotho can directly interact with human vascular smooth muscle cell, and local vascular Klotho expression involved in the occurrence and development of vascular calcification, but the mechanism is not known. DNA methylation, an epigenetic approach, is a regulation of gene expression, via CpG island in the promoter region (300-3000 BP CpG rich region of the dinucleotide) methylation silencing effects on gene expression. The purpose of this study was to explore local hypermethylation of Klotho gene and vascular calcification in patients with ESRD.Methods:We selected 30 ESRD patients undergoing artereovenias fistula plastic operation, and 10 age-matched control group undergoing coronary artery bypass graft. H.E. staining was used to observe the thickness of blood vessels. Alizarin red staining was used to determine the calcium deposition. Osteoblast specific proteins expression such as osteopontin and core binding factor alpha 1 were detected by immunohistochemical method. Vascular smooth muscle cells specific protein alpha-smooth muscle actin. Pyrosequencing assay were used to detect vascular Klotho methylation level. Klotho protein expression and serum indoxyl sulfate level were examed.Results:The average artery pressure, serum phosphorus and iPTH were higher in uremic patients than that of in control group. IMT of radial artery in uremic group was significantly higher than control group (0.68±0.53 vs 0.18±0.13 mm, P<0.05). Radial artery wall/vascular diameter ratio in uremic group was also significantly higher than that in the control group (0.34±0.19 vs 0.23±0.09, P<0.05). The serum IS level in uremic patients was higher than that of control group. The serum IS levels of 21 patients with arterial calcification was higher than that of 9 patients without arterial calcification (34.6±4.13 pg/m,29.8±4.23 pg/ml, P<0.001). Klotho methylation in uremic patients was significantly higher than control group (43.2±1.7% vs.8.7±0.8%, P<0.001); Klotho expression was positive in radial artery of 30 cases; DNMT1 expression in uremic patients was significantly higher than that in the control group. Alizarin red staining showed 21 cases vascular calcification in 30 cases of radial artery specimens, the incidence rate was 70%, the control group had no calcification (P<0.01). Immunohistochemical staining showed 26 cases (87%) with OPN positive deposition in 30 cases of radial artery, while OPN staining was negative in the control group (P<0.001); Cbf al were positive in 28 cases of radial artery in uremic patients while the control group had no positive expression; 28 cases of radial artery were negitive with a-SMA in uremic patients which were significantly lower than the control group. Pearson correlation analysis showed that the serum IS and Klotho methylation was positively (r=0.587, P<0.01); Klotho methylation and degree of arterial calcification was positively correlated (r=0.549, P< 0.01); Pearson correlation analysis showed that the serum IS was positively correlated with vascular calcification (r= 0.763, P<0.01).Conclusion:Obvious calcification exsited in uremic patients. Serum IS levels in uremic patients were significantly higher than the normal controls.Serum IS was positively correlated with the severy of vascular calcification. Klotho hypermethylation exsited in uremic patients.Part II Klotho gene hypermethylation potentiates vascular calcification induced by indoxyl sulfate in HASMCsBackground:IS is related with cardiovascular disease and all-cause mortality in ESRD patients. IS can promote vascular smooth muscle cells differentiation to osteoblast, but its mechanism is still unclear. The first part of our study confirmed that hypermethylation of Klotho gene, serum concentration of IS and vascular Klotho gene methylation is closely related to vascular calcification in ESRD patients. The purpose of this study is to to explore the role hypermethylation of Klotho gene in vascular smooth muscle cells osteoblast transformation induced by IS.Methods:The standard IS were purchersed from Sigma company. IS concentration of 0,200,500,1000 μmol/L were used. Other group were added with culture medium IS 1000 μmol/L and 0,1,10 mol/L of 5-aza-2-deoxycytidine for 6 days. Klotho methylation level were evaluated by pyrosequencing assay; Cbf a 1, osteopontin, a-SMA, Klotho, DNMT1, DNMT3a, DNMT3b were examed by Real-time PCR. Western blot was used to exame the protein expression of osteopontin, a-SMA, Klotho and DNMT1.Results:Alizarin red staining showed that IS promoted HASMCs calcification. IS induced Klotho gene hypermethylation in HASMCs. Klotho mRNA levels were decreased with the increase of IS concentration. Klotho protein also decreased with increasing the concentration of IS. OPN and Cbfal mRNA expression were increased after 6days of IS incubation, OPN expression in control group and IS group were (1.02±0.12 vs.6.11±0.68). Expression of OPN in IS group was higher than that of control group (P<0.01). After 6D incubation, Cbfal in control group and IS group (1000 mol/L) expression was (1.02±0.12 vs 5.21±1.08). a-SMA expression was significantly decreased. IS increased the expression of DNMT1. 5-aza-2-deoxycytidine reduced the degree of Klotho methylation. 5-aza-2-deoxycytidine decreased the expression of DNMT1, while calcification.were significantly reduced in HASMCs.Conclusion:IS increased Klotho methylation level in HASMCs. IS induced vascular calcification via hypermethylation of Klotho gene hypermethylation. 5-aza-2-deoxycytidine inhibited vascular calcification.via decreasing Klotho gene methylation level.Part Ⅲ Klotho gene hypermethylation promotes vascular calcification induced by indoxyl sulfate in CKD rat modelBackground:Vascular calcification is prevalent in uremic patients. Vascular Klotho gene hypermethylation and serum IS are closely related to vascular calcification. Indoxyl sulfate can induce vascular smooth muscle cells deferitiate into osteoblasts. IS can increase Klotho methylation level by inhibit the activity of DNMT1 in vascular smooth muscle cells, and decressed the expression of Klotho protein. The purpose of this study was to to explore the hypermethylation of Klotho gene in vascular calcification in uremic rats induced by IS.Methods:24 SD rats underwent 5/6 nephrectomy were randomly divided into 3 groups. Each group were administered intervention.The control group (8 rats), PBS/48h, intraperitoneal injection,24 weeks; Experimental group (8 rats), IS/100mg/Kg/48h, intraperitoneal injection,24 weeks; Experimental group (8 rats), IS/100mg/Kg/48h+5-aza-2-deoxycytidine/10mg/Kg/48h, intraperitoneal injection,24 weeks. After 24 weeks, blood and urine were collected. To observe the effect of thickness of HE staining, alizarin red staining of vascular calcification, immunohistochemistry staining of Cbf a 1, OPN, a-SMA, Klotho, DNMTl protein expression condition. Aortic specimens to extract DNA, take RNA and protein, and pathological histology paraformaldehyde treatment. Pyrosequencing detection of Klotho methylation level were measured; Real-time PCR for Cbf a 1, OPN, α-SMA, Klotho, DNMT1, DNMT3a, DNMT3b. Western blot method for the determination of Cbf a 1 of each nucleus, OPN, α-SMA, Klotho and DNMT1.Results:Alizarin red staining showed that IS promoted calcium deposition vassels. IS induced Klotho gene hypermethylation. Compared with the control group and the experimental group, IS group had Klotho gene methylation increased significantly. Control group vs IS in experimental group (29± 5% vs.6%±2) (*P<0.001). The mRNA and protein levels of Klotho in IS group were significantly decreased.The expression of DNMT1 in IS group was significantly increased. Osteopontin and Cbfal increased while a-SMA decreased in IS group than the control group. Klotho methylation level decreased significantly in IS+5-aza-2-deoxycytidine group than that in IS group. Klotho protein expression increased in IS+5-aza-2-deoxycytidine group than that in IS group, while the expression of DNMT1 protein decreased. At the same time, Alizarin red staining showed calcification decreased significantly in IS+5-aza-2-deoxycytidine group than that in IS group. Osteopontin and Cbfal protein expression decreased in IS+5-aza-2-deoxycytidine group, while the expression of a-SMA increased,showing the degree of vascular calcification significantly reduced.Conclusions:IS induced vascular calcification by hypermethylation of Klotho gene in vivo experiments.5-aza-2-deoxycytidine inhibited the hypermethylation of Klotho gene and further inhibit vascular calcification.更多还原