EPC在DR发病及治疗中作用的实验研究

发布时间：2018-02-01 00:46

本文关键词： 糖尿病视网膜病变内皮祖细胞细胞培养辛伐他汀动物实验　出处：《天津医科大学》2013年博士论文　论文类型：学位论文

【摘要】：目的：本研究通过建立Wistar大鼠糖尿病视网膜病变(diabetic retinopathy, DR)模型并检测骨髓内皮祖细胞(endothelial progenitor cells, EPC)的数量及功能变化,探讨EPC在DR发病机制中的作用：进而应用辛伐他汀动员大鼠DR模型体内EPC,观察其对大鼠DR模型视网膜病变的修复作用及机制。方法：(1)雄性Wistar大鼠50只,随机分为正常对照(CON)组(14只鼠)和糖尿病(DM)组(36只鼠)。通过腹腔注射链脲佐菌素(STZ)建立DR模型,根据病程,将DM组大鼠再分为糖尿病1个月(DM1)组、糖尿病3个月(DM3)组、糖尿病6个月(DM6)组,每组各12只。同时于各相应时间点摘除大鼠眼球行苏木精-伊红(HE)染色,采用免疫组织化学法分析血管内皮生长因子(VEGF)在大鼠视网膜中的表达,透射电子显微镜进行视网膜病理组织学检查。(2)根据前一部分DR大鼠模型,采用密度梯度离心法从各组大鼠骨髓获取单个核细胞,将其接种于纤维连接蛋白包被的培养板；培养7d后,利用Dil-acLDL和FITC-UEA-I细胞荧光染色法及流式细胞仪CD34和CD133抗体标记法鉴定EPC。在倒置相差显微镜下对EPC计数克隆形成单位(CFU),以评估骨髓EPC的数量水平；采用MTT比色法、Transwell小室和粘附能力测定实验观察EPC的增殖、迁移和粘附能力。(3)雄性Wistar大鼠80只,以随机数字表法分为正常对照组、模型对照组、安慰剂组、辛伐他汀组,每组各20只大鼠。实验组采用腹腔注射STZ建立DR大鼠模型。正常对照组、模型对照组不给予任何干预；辛伐他汀组予辛伐他汀20mg/kg灌胃,1次/d；安慰剂组给予等量蒸馏水灌胃,1次/d。分别于1、4及12周时取静脉血,采用流式细胞仪分别计数各组大鼠外周血EPC数量的变化。于12周时处死大鼠,摘除眼球行HE染色,采用伊文思蓝(EB)定量检测血-视网膜屏障(BRB)的破坏程度,免疫组织化学法分析CD31在大鼠视网膜中的表达及透射电子显微镜进行视网膜病理组织学检查。实时定量逆转录聚合酶链反应(RT-PCR)检测内皮型一氧化氮合酶(eNOS)、诱导型一氧化氮合酶(iNOS)及血管生成素-1(Ang-1)在视网膜的表达。对比分析EPC的数量改变与视网膜病理变化的相互关系。结果：(1)DM大鼠血糖明显升高,体重明显下降。DM1、DM3组视网膜视网膜厚度变薄,细胞排列紊乱,细胞核肿胀、体积增大,DM6组视网膜厚度更薄,细胞排列紊乱更加明显,部分血管扩张。DM1、DM3、DM6组视网膜VEGF表达较CON组明显增高,差异均有统计学意义。DM1组毛细血管内皮细胞及周细胞核膜轻微凹陷,异染色质聚集靠边。DM3组内皮细胞水肿,毛细血管基底膜增厚,电子密度明显增大。DM6组内皮细胞肿胀变形,基底膜增厚显著,管腔明显狭窄,甚至闭塞。(2)DM1、DM3、DM6组大鼠骨髓来源EPC-CFU数量、EPC增殖能力、迁移能力及粘附能力较CON组降低,差异均有统计学意义。(3)注射STZ后1、4、12周时安慰剂组大鼠外周血EPC计数均降低；注射STZ后1、4、12周时辛伐他汀组大鼠外周血EPC计数明显高于模型对照组和安慰剂组,差异均有统计学意义。模型对照组和安慰剂组大鼠视网膜细胞排列紊乱,内皮细胞水肿,基底膜增厚显著,电子密度增加,周细胞线粒体肿胀,嵴消失；辛伐他汀组大鼠视网膜各层组织水肿减轻,内皮细胞及周细胞核膜轻微凹陷,异染色质聚集靠边,管腔未变形。模型对照组和辛伐他汀组大鼠视网膜平均EB渗漏量较正常对照组显著增加,辛伐他汀组较模型对照组明显减少；CD31在正常对照组表达为弱阳性,辛伐他汀组表达强度较模型对照组增强；eNOS在模型对照组表达较正常对照组明显减弱,辛伐他汀组表达强度较模型对照组增强；iNOS和Ang-1在模型对照组表达较正常对照组明显增强,辛伐他汀组表达强度较模型对照组减弱,差异均有统计学意义。结论：(1)糖尿病大鼠骨髓EPC数量较正常大鼠降低,生物学功能减退。随着DR的进展,EPC数量逐渐回升。(2)辛伐他汀可动员DR大鼠外周血EPC,诱导视网膜内皮细胞迁徙分化,减缓DR进展,其可能机制为调节eNOS和iNOS等内皮形成相关因子。
[Abstract]:Objective: This study through the establishment of diabetic retinopathy in rats with Wistar (diabetic retinopathy DR) model and detection of bone marrow endothelial progenitor cells (endothelial progenitor cells, EPC) the number and function changes, to explore the role of EPC in the pathogenesis of DR and simvastatin mobilization in vivo EPC DR rat model, observe the retinal lesions the rat model of DR repair effect and mechanism.
Methods: (1) 50 male Wistar rats were randomly divided into normal control (CON) group (14 rats) and diabetes mellitus (DM) group (36 rats). By intraperitoneal injection of streptozotocin (STZ) to establish DR model, according to the course of disease, DM rats were divided into diabetes mellitus 1 months (DM1 group), diabetes for 3 months (DM3 group), diabetes for 6 months (DM6) group, 12 rats in each group. At the same time in the corresponding time points in rat with eye hematoxylin eosin (HE) staining, immunohistochemical analysis of vascular endothelial growth factor (VEGF) in the rat retina in the form of transmission electron microscopy of retinal histopathological examination. (2) according to the first part of the DR rat model to obtain mononuclear cells from the bone marrow of rats by density gradient centrifugation and inoculated on fibronectin coated culture; culture after 7d staining and flow cytometry. Fine with Dil-acLDL and FITC-UEA-I cell fluorescence CD34 and CD133 cell antibody labeling and identification of EPC. forming unit count of EPC clone under inverted phase contrast microscope (CFU), the number of level evaluation of bone marrow EPC; MTT assay was used to determine Transwell cell adhesion ability and experimental observation on EPC proliferation, migration and adhesion ability. (3) 80 Wistar male rats were randomly divided into normal control group, model control group, placebo group, simvastatin group, with 20 rats in each group. The experimental group by intraperitoneal injection of STZ DR rat model was established. The normal control group, model control group was not given any intervention; simvastatin group were given simvastatin 20mg/kg orally, 1 times /d; the placebo group were given equal volume of distilled water, 1 /d. and 1,4 respectively in 12 weeks when taking venous blood by flow cytometry were observed in rat peripheral blood EPC number changes. In 12 weeks the rats were sacrificed and the removal of eye ball by stained with HE. Evans blue (EB) quantitative detection of blood retinal barrier (BRB) damage, immunohistochemical analysis of CD31 expression in rat's retina and transmission electron microscopy of retinal histopathological examination. Real time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) detection of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase the enzyme (iNOS) and angiopoietin -1 (Ang-1) in the retina. The expression of the relationship between the number of changes compared with retinal pathological changes of EPC.
Results: (1) the blood glucose of DM rats significantly increased, weight decreased.DM1, DM3 group of retinal thinning of the retina, cell disorder, nuclear swelling, increased volume, DM6 group of retinal thickness, cell disorder is more obvious, part of.DM1 DM3 DM6, vascular dilatation, retinal VEGF expression was significantly higher than that of CON group.DM1 group, there were statistically significant differences in capillary endothelial cells and pericytes was slightly depressed, heterochromatin aggregation endothelial cells in.DM3 group by edema, capillary basement membrane thickening, the electron density of.DM6 group significantly increased endothelial cell swelling, basement membrane thickening significantly, lumen stenosis or occlusion. (2) DM1, DM3, DM6 group of rat bone marrow derived EPC-CFU, EPC proliferation, migration and adhesion ability is lower than that of CON group, the differences were statistically significant. (3) injection of STZ after 1,4,12 weeks of placebo in rat peripheral blood EP The count of C decreased; 1,4,12 weeks after injection of STZ simvastatin group rat peripheral blood EPC count was significantly higher than the model group and the placebo group, the differences were statistically significant. The model control group and the placebo group of retinal cells of rat endothelial cells arranged in disorder, edema, basement membrane thickening significantly increased electron density of mitochondria, week swelling, cristae; simvastatin group rats retinal layers of tissue edema, endothelial cells and pericytes was slightly depressed, heterochromatin aggregation aside, lumen undeformed. Model group and simvastatin group rat retinal average EB leakage was significantly increased compared with normal control group, simvastatin group than in model control group was significantly reduced in CD31; the normal control group weakly positive expression, the expression intensity of simvastatin group compared with model control group, eNOS in model group increased; expression compared with normal control group was significantly reduced The expression intensity of the simvastatin group was stronger than that of the model control group. The expression of iNOS and Ang-1 in the model control group was significantly higher than that in the normal control group, and the expression intensity in simvastatin group was lower than that in the model control group, and the difference was statistically significant.
Conclusion: (1) the number of bone marrow EPC in diabetic rats compared with normal rats decreased and the biological function. With the development of DR, the number of EPC increased gradually. (2) simvastatin can mobilize peripheral blood EPC he DR rats, induced retinal endothelial cell migration and differentiation, slowing the progression of DR, the possible mechanism of the formation of related factors for the regulation of eNOS and iNOS endothelial.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R774.1;R587.2

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