大鼠骨髓间充质干细胞转分化为胰岛样细胞及其保护移植胰岛功能的研究
发布时间：2018-11-12 07:42
【摘要】：目的： (1) 观察高血糖状态下大鼠骨髓中是否存在胰岛样细胞。 (2) 建立体外分离培养大鼠骨髓间充质干细胞(BM-MSCs)的方法，确定其生物学特性。 (3) 体外诱导BM-MSCs转分化为胰岛样细胞，，评估其生物学功能。 (4) 探讨BM-MSCs在糖尿病大鼠体内转分化为胰岛素阳性细胞的潜能。 (5) 观察BM-MSCs对移植胰岛功能的保护作用。 方法： (1) 分别采用腹腔注射链脲佐菌素(STZ)或高糖诱导Sprague-Dawley(SD)大鼠糖尿病动物模型和一过性高血糖状态，同龄正常SD大鼠作对照。经密度梯度离心法分离、贴壁法培养原代骨髓细胞，倒置显微镜下观察细胞形态，免疫荧光激光共聚焦显微镜观察胰岛相关激素的表达，RT-PCR法检测胰岛相关激素、β细胞标志(GLUT-2、GK、GLP-1R)和转录因子(PDX-1、Ngn3、NeuroD1、PAX-6、NKX2.2)基因的表达。流式细胞术检测胰岛相关激素的阳性细胞数，以及胰岛素与CD45／CD90共表达率，ELISA检测葡萄糖刺激的胰岛素分泌量(GSIS)。 (2) 采用密度梯度离心联合贴壁筛选法分离、培养SD大鼠BM-MSCs，观察细胞形态和超微结构，绘制生长曲线，测定对数生长期的细胞倍增时间，纤维母细胞集落形成实验(CFU-F)检测细胞增殖能力，流式细胞术检测细胞周期。采用免疫荧光法和流式细胞术检测细胞表面CD45／CD90、CD45／CD29表达及阳性率。并分别予成骨和成脂诱导分化，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.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R329.2;R587.1
本文编号：2326501
[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.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R329.2;R587.1
【引证文献】
相关硕士学位论文 前1条
1 何俊丹;兔骨髓间充质干细胞向胰岛细胞分化的研究[D];河南农业大学;2012年
本文编号：2326501
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