从SCAP功能失调探讨慢性肾脏病患者主要心血管疾病的病因与分子机制

发布时间：2018-05-30 18:21

本文选题：动脉培养 + 高磷血症　；参考：《重庆医科大学》2017年硕士论文

【摘要】：第一部分体外脐静脉高磷培养血管钙化模型目的:通过建立动脉体外高磷培养模型模拟体内高磷血症致血管钙化(vessels calcification,VC)过程。方法:脐静脉M199培养基无血清培养,分为正常组(Pi=1mmol/L)、高磷组(Pi=3mmol/L)。利用Von Kossa染色法和茜素红染色法对脐静脉切片进行染色,观察并比较两种方法的优缺点,HE染色对脐静脉组织形态进行评估。结果:体外血管钙化模型建模成功,高磷组钙化明显高于正常组(P0.01)。体外培养在第二十天时组织结构清晰,细胞核染色清楚,胞浆红染。对比两种钙化染色方法,Von kossa染色法对血管中层微钙化的显色比茜素红更清晰。结论:20天体外高磷(Pi=3mmol/L)培养建立血管钙化模型,选择Von kossa染色鉴定动脉中层微钙化。第二部分磷诱导动脉中层血管平滑肌细胞钙化与SCAP表达的关系目的:通过慢病毒敲除脐静脉SCAP,研究在高磷致动脉钙化体外培养模型中,SCAP蛋白对钙盐沉积的影响。方法:脐静脉自母体取出后,无血清培养2天,换液加慢病毒培养3天,加磷培养20天分为四组,对照组、高磷组、SCAP敲除组、高磷+SCAP敲除组。VK染色定量血管钙化,WB检测四组组织SCAP敲除效果和RUNX2表达情况。结果:Von kossa染色和血管钙定量结果表明体外培养的脐静脉钙化高磷组高于正常组(P0.01),高磷+SCAP蛋白敲除组高于SCAP蛋白敲除组(P0.01);western blotting结果示RUNX2蛋白在高磷组表达量明显高于对照组(P0.01),高磷+SCAP敲除组与SCAP敲除组比较RUNX2蛋白表达量显著下降(P0.05)。结论:SCAP蛋白可能参与了磷致动脉中层钙盐沉积过程,并对钙化起促进作用。第三部分血清磷通过调节SCAP蛋白过表达加速动脉粥样硬化进程目的:通过临床慢性肾病(CKD)透析患者的基本资料、桡动脉组织切片染色和脐静脉体外模拟高磷血症培养,探讨磷与动脉粥样硬化性疾病的关系。方法:按照排除纳入条件筛选CKD病例,分为动脉粥样硬化(atherosclerosis,AS)组和非动脉粥样硬化(NAS)组,用Logistic回归分析检验两组有统计学差异的指标。将收取桡动脉按照患者血清Pi水平分为高磷组和正常组,免疫荧光方法检测桡动脉中SREBPs裂解激活蛋白(SREBP Cleavage Activating Protein,SCAP)、LDL受体((LDL receptor,LDLr)、胆固醇调节原件结合蛋白2(Sterol Regulatory Element Banding Protein 2,SREBP2)和羟甲基戊二酸单酰辅酶A还原酶(Hydroxy Methylglutaryl Coenzyme A Reductase,HMGCo AR)表达情况。WB和免疫组化检测高磷培养脐静脉SCAP、LDLr、SREBP2、HMGCo AR表达量。结果:logistic回归分析结果表明,血清磷是AS的独立致病因素。桡动脉免疫荧光染色显示,高磷组SCAP、EREBP2、HMGCo AR、LDLr、RUNX2表达均高于正常组(P0.05)。WB示脐静脉高磷组与对照组比较SCAP、SREBP2、HMGCo A、LDLr均上调(P0.05),免疫组化显示结果与WB一致。结论:血清磷可能通过上调SCAP蛋白、打破SCAP-SREBP2通路的负反馈调节,导致内膜细胞内脂质蓄积从而加速动脉粥样硬化病的发生发展。
[Abstract]:Part one: vascular calcification model of umbilical vein hyperphosphorous culture in vitro objective: to simulate the process of vessel calcification induced by hyperphosphatemia in vivo by establishing an in vitro vascular calcification model. Methods: the umbilical vein M199 medium was serum-free and was divided into normal group (1 mmol / L) and high phosphorus group (3 mmol / L). Umbilical vein sections were stained with Von Kossa staining and alizarin red staining. The advantages and disadvantages of the two methods were observed and compared to evaluate the histological morphology of umbilical vein by HE staining. Results: the model of vascular calcification in vitro was successfully established, and the calcification in high phosphorus group was significantly higher than that in normal group (P 0.01). On the 20th day, the tissue structure was clear, the nucleus was stained clearly and the cytoplasm was red stained. Compared with two calcification staining methods, Von kossa staining was more clear than alizarin red in the development of microcalcification in vascular media. Conclusion the vascular calcification model was established by 20 days in vitro culture with high phosphorous content in vitro, and Von kossa staining was selected to identify the microcalcification in the arterial media. The second part is the relationship between phosphorus-induced calcification of vascular smooth muscle cells and the expression of SCAP. Objective: to investigate the effect of phosphorus-induced calcification of arterial smooth muscle cells on calcium deposition in vitro by lentivirus knockout of umbilical vein. Methods: after umbilical vein was removed from mother, serum free culture for 2 days, liquid exchange plus lentivirus culture for 3 days, phosphorus culture for 20 days were divided into four groups: control group, high phosphorus group, SCAP knockout group. High phosphorous SCAP knockout group. VK staining quantitative vascular calcification wideband detected SCAP knockout effect and RUNX2 expression in four groups. Results the expression of RUNX2 protein in high phosphorus group was significantly higher than that in SCAP protein knockout group and that in high phosphorous SCAP group was higher than that in SCAP protein knockout group. The results showed that the expression of RUNX2 protein in high phosphorus group was significantly higher than that in high phosphorus group. Compared with the SCAP knockout group, the expression of RUNX2 protein decreased significantly in the control group (P 0.01) and the high phosphorus SCAP knockout group (P 0.05). Conclusion the protein of: SCAP may be involved in the process of calcium deposition in the middle layer of artery induced by phosphorus, and may play a role in promoting calcification. The third part of serum phosphorus accelerates the process of atherosclerosis by regulating the overexpression of SCAP protein. Objective: to study the basic data of hemodialysis patients with chronic nephropathy, radial artery tissue section staining and umbilical vein culture in vitro to simulate hyperphosphatemia. To investigate the relationship between phosphorus and atherosclerotic diseases. Methods: the patients with CKD were selected according to the exclusion criteria and divided into two groups: atherosclerotic atherosclerosis group and non-atherosclerotic group. Logistic regression analysis was used to test the statistical difference between the two groups. The received radial artery was divided into high phosphorus group and normal group according to the serum Pi level of the patients. Detection of the expression of SREBPs lytic activator protein (SREBP Cleavage Activating protein), SREBPs receptor (2(Sterol Regulatory Element Banding Protein _ 2 SREBP2) and hydroxyxy Methylglutaryl Coenzyme A Reductase (HMG) in radial artery by immunofluorescence And immunohistochemistry was used to detect the expression of HMGCo AR in cultured umbilical vein SCAPP LDLrN SREBP 2 and HMGCo AR. Results the results of logistic regression analysis showed that serum phosphorus was an independent pathogenic factor of as. Radial artery immunofluorescence staining showed that the expression of RUNX2 in SCAPP EREBP2HMGCo ARN LDLrN RUNX2 in high phosphorus group was higher than that in normal control group (P 0.05). Compared with control group, SCAPP SREBP2HMGCo AMGCo ALDLr was up-regulated, and the immunohistochemical results were consistent with WB. Conclusion: serum phosphorus may accelerate the development of atherosclerotic disease by up-regulating the SCAP protein and breaking the negative feedback regulation of SCAP-SREBP2 pathway, resulting in the accumulation of lipid in intimal cells.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692;R54

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