本文關(guān)鍵詞： 糖尿病視網(wǎng)膜病變 內(nèi)皮祖細(xì)胞 細(xì)胞培養(yǎng) 辛伐他汀 動(dòng)物實(shí)驗(yàn)　出處：《天津醫(yī)科大學(xué)》2013年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的：本研究通過(guò)建立Wistar大鼠糖尿病視網(wǎng)膜病變(diabetic retinopathy, DR)模型并檢測(cè)骨髓內(nèi)皮祖細(xì)胞(endothelial progenitor cells, EPC)的數(shù)量及功能變化,探討EPC在DR發(fā)病機(jī)制中的作用：進(jìn)而應(yīng)用辛伐他汀動(dòng)員大鼠DR模型體內(nèi)EPC,觀察其對(duì)大鼠DR模型視網(wǎng)膜病變的修復(fù)作用及機(jī)制。 方法：(1)雄性Wistar大鼠50只,隨機(jī)分為正常對(duì)照(CON)組(14只鼠)和糖尿病(DM)組(36只鼠)。通過(guò)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(gè)月(DM1)組、糖尿病3個(gè)月(DM3)組、糖尿病6個(gè)月(DM6)組,每組各12只。同時(shí)于各相應(yīng)時(shí)間點(diǎn)摘除大鼠眼球行蘇木精-伊紅(HE)染色,采用免疫組織化學(xué)法分析血管內(nèi)皮生長(zhǎng)因子(VEGF)在大鼠視網(wǎng)膜中的表達(dá),透射電子顯微鏡進(jìn)行視網(wǎng)膜病理組織學(xué)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(gè)核細(xì)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(xì)胞熒光染色法及流式細(xì)胞儀CD34和CD133抗體標(biāo)記法鑒定EPC。在倒置相差顯微鏡下對(duì)EPC計(jì)數(shù)克隆形成單位(CFU),以評(píng)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(cè)定實(shí)驗(yàn)觀察EPC的增殖、遷移和粘附能力。(3)雄性Wistar大鼠80只,以隨機(jī)數(shù)字表法分為正常對(duì)照組、模型對(duì)照組、安慰劑組、辛伐他汀組,每組各20只大鼠。實(shí)驗(yàn)組采用腹腔注射STZ建立DR大鼠模型。正常對(duì)照組、模型對(duì)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(shí)取靜脈血,采用流式細(xì)胞儀分別計(jì)數(shù)各組大鼠外周血EPC數(shù)量的變化。于12周時(shí)處死大鼠,摘除眼球行HE染色,采用伊文思藍(lán)(EB)定量檢測(cè)血-視網(wǎng)膜屏障(BRB)的破壞程度,免疫組織化學(xué)法分析CD31在大鼠視網(wǎng)膜中的表達(dá)及透射電子顯微鏡進(jìn)行視網(wǎng)膜病理組織學(xué)檢查。實(shí)時(shí)定量逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)檢測(cè)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(dá)。對(duì)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(xì)胞排列紊亂,細(xì)胞核腫脹、體積增大,DM6組視網(wǎng)膜厚度更薄,細(xì)胞排列紊亂更加明顯,部分血管擴(kuò)張。DM1、DM3、DM6組視網(wǎng)膜VEGF表達(dá)較CON組明顯增高,差異均有統(tǒng)計(jì)學(xué)意義。DM1組毛細(xì)血管內(nèi)皮細(xì)胞及周細(xì)胞核膜輕微凹陷,異染色質(zhì)聚集靠邊。DM3組內(nèi)皮細(xì)胞水腫,毛細(xì)血管基底膜增厚,電子密度明顯增大。DM6組內(nèi)皮細(xì)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(lái)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(jì)學(xué)意義。(3)注射STZ后1、4、12周時(shí)安慰劑組大鼠外周血EPC計(jì)數(shù)均降低；注射STZ后1、4、12周時(shí)辛伐他汀組大鼠外周血EPC計(jì)數(shù)明顯高于模型對(duì)照組和安慰劑組,差異均有統(tǒng)計(jì)學(xué)意義。模型對(duì)照組和安慰劑組大鼠視網(wǎng)膜細(xì)胞排列紊亂,內(nèi)皮細(xì)胞水腫,基底膜增厚顯著,電子密度增加,周細(xì)胞線(xiàn)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(xì)胞及周細(xì)胞核膜輕微凹陷,異染色質(zhì)聚集靠邊,管腔未變形。模型對(duì)照組和辛伐他汀組大鼠視網(wǎng)膜平均EB滲漏量較正常對(duì)照組顯著增加,辛伐他汀組較模型對(duì)照組明顯減少；CD31在正常對(duì)照組表達(dá)為弱陽(yáng)性,辛伐他汀組表達(dá)強(qiáng)度較模型對(duì)照組增強(qiáng)；eNOS在模型對(duì)照組表達(dá)較正常對(duì)照組明顯減弱,辛伐他汀組表達(dá)強(qiáng)度較模型對(duì)照組增強(qiáng)；iNOS和Ang-1在模型對(duì)照組表達(dá)較正常對(duì)照組明顯增強(qiáng),辛伐他汀組表達(dá)強(qiáng)度較模型對(duì)照組減弱,差異均有統(tǒng)計(jì)學(xué)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(xué)功能減退。隨著DR的進(jìn)展,EPC數(shù)量逐漸回升。(2)辛伐他汀可動(dòng)員DR大鼠外周血EPC,誘導(dǎo)視網(wǎng)膜內(nèi)皮細(xì)胞遷徙分化,減緩DR進(jìn)展,其可能機(jī)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(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R774.1;R587.2

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