【摘要】：目的： (1) 觀察高血糖狀態(tài)下大鼠骨髓中是否存在胰島樣細(xì)胞。 (2) 建立體外分離培養(yǎng)大鼠骨髓間充質(zhì)干細(xì)胞(BM-MSCs)的方法，確定其生物學(xué)特性。 (3) 體外誘導(dǎo)BM-MSCs轉(zhuǎn)分化為胰島樣細(xì)胞，，評(píng)估其生物學(xué)功能。 (4) 探討B(tài)M-MSCs在糖尿病大鼠體內(nèi)轉(zhuǎn)分化為胰島素陽性細(xì)胞的潛能。 (5) 觀察BM-MSCs對(duì)移植胰島功能的保護(hù)作用。 方法： (1) 分別采用腹腔注射鏈脲佐菌素(STZ)或高糖誘導(dǎo)Sprague-Dawley(SD)大鼠糖尿病動(dòng)物模型和一過性高血糖狀態(tài)，同齡正常SD大鼠作對(duì)照。經(jīng)密度梯度離心法分離、貼壁法培養(yǎng)原代骨髓細(xì)胞，倒置顯微鏡下觀察細(xì)胞形態(tài)，免疫熒光激光共聚焦顯微鏡觀察胰島相關(guān)激素的表達(dá)，RT-PCR法檢測(cè)胰島相關(guān)激素、β細(xì)胞標(biāo)志(GLUT-2、GK、GLP-1R)和轉(zhuǎn)錄因子(PDX-1、Ngn3、NeuroD1、PAX-6、NKX2.2)基因的表達(dá)。流式細(xì)胞術(shù)檢測(cè)胰島相關(guān)激素的陽性細(xì)胞數(shù)，以及胰島素與CD45／CD90共表達(dá)率，ELISA檢測(cè)葡萄糖刺激的胰島素分泌量(GSIS)。 (2) 采用密度梯度離心聯(lián)合貼壁篩選法分離、培養(yǎng)SD大鼠BM-MSCs，觀察細(xì)胞形態(tài)和超微結(jié)構(gòu)，繪制生長(zhǎng)曲線，測(cè)定對(duì)數(shù)生長(zhǎng)期的細(xì)胞倍增時(shí)間，纖維母細(xì)胞集落形成實(shí)驗(yàn)(CFU-F)檢測(cè)細(xì)胞增殖能力，流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期。采用免疫熒光法和流式細(xì)胞術(shù)檢測(cè)細(xì)胞表面CD45／CD90、CD45／CD29表達(dá)及陽性率。并分別予成骨和成脂誘導(dǎo)分化，Von Kossa染
[Abstract]:Objective: (1) to observe whether islet like cells exist in bone marrow of rats with hyperglycemia. (2) to establish a method to isolate and culture rat bone marrow mesenchymal stem cells (BM-MSCs) in vitro and determine its biological characteristics. (3) BM-MSCs was induced to differentiate into islet like cells in vitro and its biological function was evaluated. (4) to investigate the potential of BM-MSCs to differentiate into insulin-positive cells in diabetic rats. (5) to observe the protective effect of BM-MSCs on islet transplantation. Methods: (1) Diabetic model and transient hyperglycemia of Sprague-Dawley (SD) rats were induced by intraperitoneal injection of streptozotocin (STZ) or hyperglycemia respectively, and normal SD rats of the same age were used as control. Primary bone marrow cells were isolated by density gradient centrifugation, primary bone marrow cells were cultured by adherent method, cell morphology was observed under inverted microscope, expression of islet related hormones was observed by immunofluorescence confocal laser microscope, islet related hormones were detected by RT-PCR method. Expression of 尾-cell marker (GLUT-2,GK,GLP-1R) and transcription factor (PDX-1,Ngn3,NeuroD1,PAX-6,NKX2.2) gene. Flow cytometry was used to detect the number of islet related hormone positive cells and the coexpression rate of insulin and CD45/CD90. ELISA was used to detect glucose-stimulated insulin secretion (GSIS). (2) SD rats were isolated by density gradient centrifugation combined with adherent screening method. The morphology and ultrastructure of BM-MSCs, were observed, the growth curve was drawn, and the cell doubling time of logarithmic growth period was measured. Fibroblast colony forming assay (CFU-F) was used to detect cell proliferation and flow cytometry was used to detect cell cycle. The expression and positive rate of CD45/CD90,CD45/CD29 on cell surface were detected by immunofluorescence and flow cytometry. Osteogenic and adipogenic differentiation were stained with, Von Kossa.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R329.2;R587.1

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 何俊丹;兔骨髓間充質(zhì)干細(xì)胞向胰島細(xì)胞分化的研究[D];河南農(nóng)業(yè)大學(xué);2012年

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