本文關(guān)鍵詞： 糖尿病視網(wǎng)膜病變 內(nèi)皮祖細胞 細胞培養(yǎng) 辛伐他汀 動物實驗　出處：《天津醫(yī)科大學》2013年博士論文　論文類型：學位論文

【摘要】：目的：本研究通過建立Wistar大鼠糖尿病視網(wǎng)膜病變(diabetic retinopathy, DR)模型并檢測骨髓內(nèi)皮祖細胞(endothelial progenitor cells, EPC)的數(shù)量及功能變化,探討EPC在DR發(fā)病機制中的作用：進而應(yīng)用辛伐他汀動員大鼠DR模型體內(nèi)EPC,觀察其對大鼠DR模型視網(wǎng)膜病變的修復(fù)作用及機制。 方法：(1)雄性Wistar大鼠50只,隨機分為正常對照(CON)組(14只鼠)和糖尿病(DM)組(36只鼠)。通過腹腔注射鏈脲佐菌素(STZ)建立DR模型,根據(jù)病程,將DM組大鼠再分為糖尿病1個月(DM1)組、糖尿病3個月(DM3)組、糖尿病6個月(DM6)組,每組各12只。同時于各相應(yīng)時間點摘除大鼠眼球行蘇木精-伊紅(HE)染色,采用免疫組織化學法分析血管內(nèi)皮生長因子(VEGF)在大鼠視網(wǎng)膜中的表達,透射電子顯微鏡進行視網(wǎng)膜病理組織學檢查。(2)根據(jù)前一部分DR大鼠模型,采用密度梯度離心法從各組大鼠骨髓獲取單個核細胞,將其接種于纖維連接蛋白包被的培養(yǎng)板；培養(yǎng)7d后,利用Dil-acLDL和FITC-UEA-I細胞熒光染色法及流式細胞儀CD34和CD133抗體標記法鑒定EPC。在倒置相差顯微鏡下對EPC計數(shù)克隆形成單位(CFU),以評估骨髓EPC的數(shù)量水平；采用MTT比色法、Transwell小室和粘附能力測定實驗觀察EPC的增殖、遷移和粘附能力。(3)雄性Wistar大鼠80只,以隨機數(shù)字表法分為正常對照組、模型對照組、安慰劑組、辛伐他汀組,每組各20只大鼠。實驗組采用腹腔注射STZ建立DR大鼠模型。正常對照組、模型對照組不給予任何干預(yù)；辛伐他汀組予辛伐他汀20mg/kg灌胃,1次/d；安慰劑組給予等量蒸餾水灌胃,1次/d。分別于1、4及12周時取靜脈血,采用流式細胞儀分別計數(shù)各組大鼠外周血EPC數(shù)量的變化。于12周時處死大鼠,摘除眼球行HE染色,采用伊文思藍(EB)定量檢測血-視網(wǎng)膜屏障(BRB)的破壞程度,免疫組織化學法分析CD31在大鼠視網(wǎng)膜中的表達及透射電子顯微鏡進行視網(wǎng)膜病理組織學檢查。實時定量逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)檢測內(nèi)皮型一氧化氮合酶(eNOS)、誘導(dǎo)型一氧化氮合酶(iNOS)及血管生成素-1(Ang-1)在視網(wǎng)膜的表達。對比分析EPC的數(shù)量改變與視網(wǎng)膜病理變化的相互關(guān)系。 結(jié)果：(1)DM大鼠血糖明顯升高,體重明顯下降。DM1、DM3組視網(wǎng)膜視網(wǎng)膜厚度變薄,細胞排列紊亂,細胞核腫脹、體積增大,DM6組視網(wǎng)膜厚度更薄,細胞排列紊亂更加明顯,部分血管擴張。DM1、DM3、DM6組視網(wǎng)膜VEGF表達較CON組明顯增高,差異均有統(tǒng)計學意義。DM1組毛細血管內(nèi)皮細胞及周細胞核膜輕微凹陷,異染色質(zhì)聚集靠邊。DM3組內(nèi)皮細胞水腫,毛細血管基底膜增厚,電子密度明顯增大。DM6組內(nèi)皮細胞腫脹變形,基底膜增厚顯著,管腔明顯狹窄,甚至閉塞。(2)DM1、DM3、DM6組大鼠骨髓來源EPC-CFU數(shù)量、EPC增殖能力、遷移能力及粘附能力較CON組降低,差異均有統(tǒng)計學意義。(3)注射STZ后1、4、12周時安慰劑組大鼠外周血EPC計數(shù)均降低；注射STZ后1、4、12周時辛伐他汀組大鼠外周血EPC計數(shù)明顯高于模型對照組和安慰劑組,差異均有統(tǒng)計學意義。模型對照組和安慰劑組大鼠視網(wǎng)膜細胞排列紊亂,內(nèi)皮細胞水腫,基底膜增厚顯著,電子密度增加,周細胞線粒體腫脹,嵴消失；辛伐他汀組大鼠視網(wǎng)膜各層組織水腫減輕,內(nèi)皮細胞及周細胞核膜輕微凹陷,異染色質(zhì)聚集靠邊,管腔未變形。模型對照組和辛伐他汀組大鼠視網(wǎng)膜平均EB滲漏量較正常對照組顯著增加,辛伐他汀組較模型對照組明顯減少；CD31在正常對照組表達為弱陽性,辛伐他汀組表達強度較模型對照組增強；eNOS在模型對照組表達較正常對照組明顯減弱,辛伐他汀組表達強度較模型對照組增強；iNOS和Ang-1在模型對照組表達較正常對照組明顯增強,辛伐他汀組表達強度較模型對照組減弱,差異均有統(tǒng)計學意義。 結(jié)論：(1)糖尿病大鼠骨髓EPC數(shù)量較正常大鼠降低,生物學功能減退。隨著DR的進展,EPC數(shù)量逐漸回升。(2)辛伐他汀可動員DR大鼠外周血EPC,誘導(dǎo)視網(wǎng)膜內(nèi)皮細胞遷徙分化,減緩DR進展,其可能機制為調(diào)節(jié)eNOS和iNOS等內(nèi)皮形成相關(guān)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.

【學位授予單位】：天津醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R774.1;R587.2

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