海拔对2型糖尿病患者外周血内皮祖细胞数量及单核细胞功能的影响

发布时间：2018-07-16 17:33

【摘要】：目的：随着我国经济的发展,人群膳食结构由以植物性食物为主的膳食模式转变为动物性食物所占比例较高的膳食模式,近年来糖尿病的发病率逐年升高,而糖尿病引起的血管病变则成为糖尿病患者死亡和伤残的主要原因。循环内皮祖细胞是指从骨髓动员,能募集至损伤部位,增殖分化为成熟的内皮细胞,并融入内皮细胞层,并通过旁分泌多种血管内皮生长因子,促进血管生成的一种细胞。其在生理条件下促血管生成,以及病理条件下促进血管修复中至关重要。利用Ficoll密度梯度离心法从外周血中获得单个核细胞,然后通过血管内皮生长因子(VEGF),碱性成纤维细胞生长因子(bFGF)诱导和预涂纤连蛋白促使细胞贴壁并向内皮祖细胞分化,通过贴壁筛选可获得内皮祖细胞。研究表明,糖尿病患者外周血内皮祖细胞数量减少,功能异常,导致其血管修复和再生异常,因此内皮祖细胞和单核细胞的移植及活化则成为糖尿病血管并发症有效的治疗方法。内皮祖细胞发挥其修复损伤血管和促进血管新生的功能,受多方面因素的调解,损伤部位缺血缺氧可刺激低氧诱导因子1α (HIF-1 a)的表达,后者可激活其下游基因如基质细胞衍生因子1(SDF-1)、VEGF等的表达,促使EPCs动员,迁移至损伤部位修复损伤的血管内皮和促进血管重生。而对于低氧低气压的高海拔地区的糖尿病患者,与低海拔地区糖尿病患者相较,其外周血单核细胞功能与内皮祖细胞数量有何变化呢?因此,本文旨在分析海拔对于2型糖尿病患者内皮祖细胞数量及单核细胞功能的影响,从而为高海拔地区2型糖尿病患者血管并发症的治疗提供依据。方法：1.海拔对2型糖尿病患者外周血内皮祖细胞的影响。样本来自海拔386米的咸阳市和海拔1520米的兰州市,男女各半,均为当地40岁以上的常住汉族居民,分别为低海拔组和高海拔组,低海拔组包括2型糖尿病患者25例,健康者20例,高海拔组包括,2型糖尿病患者29例,健康者20例。采集其外周血8m1,检测其血脂、血糖、糖化血红蛋白浓度。流式细胞仪对外周血EPCs检测并计数。2.海拔对2型糖尿病患者外周血单核细胞功能的影响。酶联免疫吸附实验(ELISA)检测血清中HIF-1α, SDF-1、IL-8、VEGF、HCY的浓度。密度梯度离心法获取外周血单个核细胞,荧光定量PCR检测单核细胞LFA-1、CXCR2、CXCR4、 CXCR7的基因表达。蛋白免疫印迹(WesternBlot)检测单核细胞HIF-1 α CXCR2. CXCR4、CXCR7的蛋白表达。结果：1.海拔对2型糖尿病患者外周血内皮祖细胞的影响。与健康者相比,糖尿病患者体质指数(BMI)、腰臀比(WHR)、甘油三酯、血糖及糖化血红蛋白均高于健康组；高、低海拔组2型糖尿病患者外周血EPCs均低于健康者。与低海拔组相较,高海拔地区糖尿病组和健康组外周血EPCs均低于低海拔地区的糖尿病组和健康组。将糖尿病组分为：糖尿病伴微血管并发症组、糖尿病伴大血管并发症组、糖尿病无血管并发症组,与健康组相比,其外周血EPCs的数量依次为健康者2型糖尿病无血管并发症者2型糖尿病伴微血管并发症者=2型糖尿病伴大血管并发症者。2.海拔对2型糖尿病患者外周血单核细胞功能的影响。①与健康组相比,高海拔组的T2DM患者单核细胞的HIF-1α,SDF-1, VEGF的血清浓度均较低,P0.05,IL-8的浓度无差异；低海拔健康组与T2DM组间HIF-1α,SDF-1, VEGF,IL-8的浓度均无差异。与低海拔组相比,高海拔健康组和糖尿病组的HIF-1a,SDF-1血清浓度显著升高,而VEGF和IL-8的血清浓度显著降低,P0.01。②基因表达方面,与健康者相比,低海拔T2DM组CXCR2的表达显著下调P0.001,LFA-1、CXCR4和CXCR7表达无差异；而高海拔T2DM组的CXCR2和CXCR4较健康组均显著下调,P0.001；与低海拔组相较,高海拔健康组和T2DM组的CXCR2、 CXCR4和CXCR7表达均下调,P0.05,高海拔健康组的CXCR2、CXCR4、CXCR7和LFA-1分别是低海拔组的0.1、0.67、0.43和1.5倍,LFA-1的表达上调；高海拔T2DM组的CXCR2、CXCR4、CXCR7和LFA-1的表达分别是低海拔组的0.54、0.26、0.53和1.4倍,LFA-1的表达上调,P0.05。③蛋白表达水平。与健康者相比,T2DM组的HIF-1αCXCR2、CXCR4、CXCR7的表达均降低,与低海拔地区健康组和T2DM组比较,高海拔健康组和T2DM组HIF-1α表达上调,而CXCR2、 CXCR4、CXCR7的表达显著下调。结论：在低于1500米海拔的地区,随着海拔的升高,2型糖尿病患者外周血EPCs数量减少,外周血单个核细胞的黏附性增强,而迁移及促血管形成的能力减弱。
[Abstract]:Objective: with the development of China's economy, the dietary pattern of the population is transformed from a plant based diet to a high proportion of animal food. In recent years, the incidence of diabetes has increased year by year, and the vascular disease caused by diabetes has become the main cause of death and disability in diabetic patients. Progenitor cells refer to the mobilization of the bone marrow, which can be recruited to the injured part, proliferate and differentiate into mature endothelial cells, and integrate into the endothelial cell layer, and promote vascular generation by secreting a variety of vascular endothelial growth factors by paracrine, which is essential to promote vascular repair under physiological conditions and to promote vascular repair under pathological conditions. Ficoll density gradient centrifugation obtains mononuclear cells from peripheral blood, then through vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) inducing and precoating fibronectin to induce cell adhesion and differentiation to endothelial progenitor cells. Endothelial progenitor cells can be obtained through adherence screening. Endothelial progenitor cells and monocyte transplantation and activation are effective methods for the treatment of diabetic vascular complications. Endothelial progenitor cells play a role in repairing damaged blood vessels and promoting new vascular functions, mediated by multiple factors, and injury department. Ischemic anoxia stimulates the expression of hypoxia inducible factor 1 alpha (HIF-1 a), which activates the expression of its downstream genes, such as stromal cell derived factor 1 (SDF-1), VEGF and so on, prompting EPCs mobilization, migrating to the injured site to repair damaged vascular endothelium and promoting vascular rebirth. What is the change in the number of peripheral blood mononuclear cells and endothelial progenitor cells in the peripheral blood mononuclear cells in low altitude areas? Therefore, the aim of this paper is to analyze the effect of altitude on the number of endothelial progenitor cells and the function of monocyte in type 2 diabetic patients, so as to provide the basis for the treatment of vascular complications of patients with type 2 glycan disease at high altitude. Method: the effect of 1. altitude on peripheral blood endothelial progenitor cells in type 2 diabetic patients. The samples were from Xianyang city of 386 meters above sea level and Lanzhou city of 1520 meters above sea level. Half and half of men and women were resident Han residents over 40 years old, respectively, low altitudes and high altitude groups, 25 cases of type 2 diabetes mellitus, 20 cases of healthy people, high altitudes in low altitude group. The group included 29 patients with type 2 diabetes and 20 healthy persons. The peripheral blood 8m1 was collected to detect the blood lipid, blood sugar, glycated hemoglobin concentration. Flow cytometry was used to detect the peripheral blood EPCs and the effect of.2. elevation on the function of peripheral blood mononuclear cells in type 2 diabetic patients. The enzyme linked immunosorbent assay (ELISA) was used to detect HIF-1 alpha, SDF-1, IL-8 in serum. The concentration of VEGF, HCY. Density gradient centrifugation was used to obtain peripheral blood mononuclear cells, and fluorescence quantitative PCR was used to detect the gene expression of LFA-1, CXCR2, CXCR4, CXCR7. Protein immunoblotting (WesternBlot) was used to detect HIF-1 alpha CXCR2. CXCR4, CXCR7 protein expression. Fruit: 1. altitude on peripheral blood endothelial progenitor cells of type 2 diabetic patients Compared with healthy people, the body mass index (BMI), waist to hip ratio (WHR), triglyceride, glycemic and glycated hemoglobin were higher than those in the healthy group, and the peripheral blood EPCs of the patients with type 2 diabetes in the low altitude group was lower than that of the healthy ones. Compared with the low altitude group, the EPCs of the diabetic group and the healthy group were lower than the low altitude. Diabetes group and health group were divided into diabetes group with microvascular complication group, diabetes with large blood vessel complication group and diabetic non vascular complication group. Compared with health group, the number of peripheral blood EPCs in peripheral blood was the =2 type sugar of type 2 diabetic patients with type 2 diabetes with microvascular complications. The effect of.2. elevation on peripheral blood mononuclear cell function in type 2 diabetic patients with urinary disease and great vascular complication. (1) the concentration of HIF-1 a, SDF-1, VEGF in the monocytes of T2DM patients in high altitude group were lower than those in the healthy group, and the concentration of P0.05, IL-8 was not different, and the concentration of HIF-1 alpha, SDF-1, VEGF, IL-8, between the low altitude health group and the T2DM group. Compared with the low altitude group, the serum concentration of HIF-1a and SDF-1 in the high altitude health group and the diabetic group increased significantly, while the serum concentration of VEGF and IL-8 decreased significantly. The expression of VEGF and IL-8 was significantly lower in the T2DM group at low altitude than in the healthy persons. The expression of CXCR2 in the low altitude T2DM group was significantly lower than that of P0.001, LFA-1, CXCR4 and CXCR7, while the high altitude T2DM was not different. The CXCR2 and CXCR4 of the group were significantly lower than those in the healthy group, P0.001. Compared with the low altitude group, the CXCR2, CXCR4 and CXCR7 expressions in the high altitude health group and the T2DM group were all downregulated, P0.05, and the CXCR2, CXCR4, CXCR7 and LFA-1 in the high altitude Health Group were 1.5 times as high as those in the low altitude group. The expression of XCR7 and LFA-1 were 0.54,0.26,0.53 and 1.4 times of the low altitude group, the expression of LFA-1 was up and P0.05. protein expression level. Compared with healthy people, the expression of HIF-1 alpha CXCR2, CXCR4 and CXCR7 in the T2DM group decreased. The expression of XCR7 was significantly reduced. Conclusion: in the area below 1500 m, the number of EPCs in peripheral blood of patients with type 2 diabetes decreased, and the adhesion of peripheral blood mononuclear cells increased, and the ability to migrate and promote angiogenesis was weakened.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.2

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