体外分离培养MSCs定向诱导为胰岛素分泌细胞
发布时间：2018-08-28 10:28
【摘要】： 目的研究成人骨髓间充质干细胞(MSCs)的体外分离培养及向胰岛素分泌细胞的定向诱导分化和鉴定方法,为糖尿病的治疗提供种子细胞来源。方法1.分别采用全骨髓培养法与Percoll密度梯度离心法从成人骨髓中分离MSCs,相同条件下体外培养,比较两种方法所获得的贴壁细胞克隆数、细胞形态、细胞表面标志及向脂肪细胞的分化情况。2.将全骨髓培养法获得的MSCs体外培养传代,取3～5代细胞先后予以2-巯基乙醇、谷氨酰胺、表皮生长因子(EGF)、碱性成纤维细胞生长因子(bFGF)及尼克酰胺等,通过三阶段向胰岛样细胞定向诱导分化。3.相差显微镜下观察MSCs在诱导前后的形态变化、免疫荧光鉴定胰十二指肠同源异型盒基因(PDX-1)的表达、双硫腙染色鉴定胰岛β样细胞团、电化学发光法测定胰岛素的分泌量。结果1.全骨髓培养法获得的贴壁细胞克隆数明显多于Percoll密度梯度离心法(P＜0.05)。两种方法获得的人MSCs形态无明显差异,CD44阳性表达率和CD34阴性表达率差异无统计学意义(P＞0.05),经地塞米松、胰岛素诱导后均可分化为脂肪细胞。2.MSCs经2-巯基乙醇、尼克酰胺等诱导后,细胞逐渐变圆,诱导二阶段细胞表达PDX-1,诱导三阶段形成胰岛样细胞团,双硫腙染色阳性,葡萄糖刺激有胰岛素释放;而未经诱导的MSCs呈长梭形贴壁生长,无PDX-1表达,双硫腙染色阴性,无胰岛素释放。结论1.与Percoll密度梯度离心法比较,全骨髓培养法是更为简便、实用的MSCs体外分离方法之一。2.2-巯基乙醇、谷氨酰胺、EGF、bFGF及尼克酰胺等可在体外诱导成人MSCs转分化为胰岛素分泌细胞。
[Abstract]:Objective to study the methods of isolation and culture of adult bone marrow mesenchymal stem cells (MSCs) in vitro and their differentiation into insulin secreting cells in order to provide seed cells for the treatment of diabetes mellitus. Method 1. Whole bone marrow culture method and Percoll density gradient centrifugation method were used to isolate MSCs, from adult bone marrow in vitro. The number and morphology of adherent cells obtained by the two methods were compared. Cell surface markers and differentiation into adipocytes. The MSCs obtained by whole bone marrow culture was subcultured in vitro. The cells of 5 passages were treated with 2-mercaptoethanol, glutamine, epidermal growth factor (EGF),) basic fibroblast growth factor (bFGF) and nicotinamide, respectively. The differentiation of islet like cells was induced by three stages. The morphologic changes of MSCs before and after induction were observed under phase contrast microscope, the expression of PDX-1 in pancreaticoduodenal homologous box (PDX-1) was detected by immunofluorescence, the 尾 -like cell cluster of pancreatic islet was identified by dithizone staining, and the secretion of insulin was determined by electrochemiluminescence. Result 1. The number of adherent cells obtained by whole bone marrow culture was significantly higher than that by Percoll density gradient centrifugation (P < 0. 05). There was no significant difference in positive expression rate of CD44 and negative expression rate of CD34 between the two methods (P > 0. 05). After induced by dexamethasone and insulin, human MSCs could differentiate into adipocytes. 2. MSCs were differentiated into adipocytes by 2-mercaptoethanol. After the induction of nicotinamide, the cells gradually became round, PDX-1, was induced to form islet like cell mass in three stages, dithizone staining was positive, glucose stimulated insulin release, while the uninduced MSCs was fusiform adherent growth. No PDX-1 expression, no dithizone staining, no insulin release. Conclusion 1. Compared with Percoll density gradient centrifugation, the whole bone marrow culture method is more convenient. One of the practical methods of isolation of MSCs in vitro. 2.2-mercaptoethanol, Glutamine EGFN bFGF and nicotinamide can induce adult MSCs to differentiate into insulin-producing cells in vitro.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329
本文编号：2209121
[Abstract]:Objective to study the methods of isolation and culture of adult bone marrow mesenchymal stem cells (MSCs) in vitro and their differentiation into insulin secreting cells in order to provide seed cells for the treatment of diabetes mellitus. Method 1. Whole bone marrow culture method and Percoll density gradient centrifugation method were used to isolate MSCs, from adult bone marrow in vitro. The number and morphology of adherent cells obtained by the two methods were compared. Cell surface markers and differentiation into adipocytes. The MSCs obtained by whole bone marrow culture was subcultured in vitro. The cells of 5 passages were treated with 2-mercaptoethanol, glutamine, epidermal growth factor (EGF),) basic fibroblast growth factor (bFGF) and nicotinamide, respectively. The differentiation of islet like cells was induced by three stages. The morphologic changes of MSCs before and after induction were observed under phase contrast microscope, the expression of PDX-1 in pancreaticoduodenal homologous box (PDX-1) was detected by immunofluorescence, the 尾 -like cell cluster of pancreatic islet was identified by dithizone staining, and the secretion of insulin was determined by electrochemiluminescence. Result 1. The number of adherent cells obtained by whole bone marrow culture was significantly higher than that by Percoll density gradient centrifugation (P < 0. 05). There was no significant difference in positive expression rate of CD44 and negative expression rate of CD34 between the two methods (P > 0. 05). After induced by dexamethasone and insulin, human MSCs could differentiate into adipocytes. 2. MSCs were differentiated into adipocytes by 2-mercaptoethanol. After the induction of nicotinamide, the cells gradually became round, PDX-1, was induced to form islet like cell mass in three stages, dithizone staining was positive, glucose stimulated insulin release, while the uninduced MSCs was fusiform adherent growth. No PDX-1 expression, no dithizone staining, no insulin release. Conclusion 1. Compared with Percoll density gradient centrifugation, the whole bone marrow culture method is more convenient. One of the practical methods of isolation of MSCs in vitro. 2.2-mercaptoethanol, Glutamine EGFN bFGF and nicotinamide can induce adult MSCs to differentiate into insulin-producing cells in vitro.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329
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