骨髓基质干细胞诱导分化为胰腺β细胞的研究

发布时间：2018-08-20 16:07

【摘要】：目的: 以大鼠为研究对象,探讨(1) 大鼠骨髓基质干细胞(MSCs)体外培养、定向诱导分化为胰腺β细胞;(2)大鼠骨髓基质干细胞体内分化为胰腺β细胞;(3)大鼠骨髓基质干细胞移植对糖尿病大鼠血糖的影响。方法: (1) 从大鼠股骨处取骨髓,用Ficoll 细胞分离液分离骨髓基质干细胞,贴壁培养。在细胞培养液中分别加入转化生长因子β(TGFβ)、成纤维细胞生长因子(bFGF)、烟酰胺(Nicotinamide)以及与胰岛细胞共培养,诱导骨髓基质干细胞向胰腺β细胞分化,通过细胞形态学的观察、荧光免疫组化检测胰岛素、逆转录-聚合酶链式反应(RT-PCR)检测胰岛素、生长抑素、胰高糖素和Pdx-1 胰腺相关基因mRNA的表达,判断骨髓基质干细胞是否分化为胰腺β细胞。 (2) 应用Hoechst 33258 细胞核荧光染料标记骨髓基质干细胞,将标记后的骨髓基质干细胞移植至大鼠胰腺组织中,每三天取大鼠胰腺组织做冰冻切片,观察荧光标记的细胞在组织中的定植情况,并通过荧光免疫组化检测荧光细胞胰岛素的表达,判断骨髓基质干细胞在体内是否分化为胰腺β细胞。 (3)运用单次腹腔注射链脲菌素(Streptozotocin,STZ)制备大鼠糖尿病模型,大鼠血糖连续三次高于16mmol 定为糖尿病大鼠,糖尿病大鼠随机分为两组:骨髓基质干细胞移植组和培养基注射组。将10~6骨髓基质干细胞悬浮于200μl 无血清培养液中,开腹后经微量注射器直接注射至胰腺组织中;培养基注射组作为对照组,将200μl 无血清培养液注射至胰腺组织中。分别检测细胞移植后3 天,一周大鼠血糖水平,观察大鼠血糖变化。结果: (1)大鼠骨髓基质干细胞在TGFβ细胞因子作用下,有极少数细胞分化为胰岛素蛋白水平表达阳性,胰岛素和Pdx-1 基因表达阳性的类胰腺β细胞,而生长抑素、胰高糖素基因表达为阴性;骨髓基质干细胞与胰岛细胞共培养,骨髓基质干细胞分化
[Abstract]:Objective: to investigate the culture of rat bone marrow stromal cells (MSCs) in vitro. Differentiation into pancreatic 尾 cells; (2) differentiation of rat bone marrow stromal cells into pancreatic 尾 cells in vivo; (3) effects of transplantation of rat bone marrow stromal cells on blood glucose in diabetic rats. Methods: (1) Bone marrow stromal cells were isolated from rat femur and cultured with Ficoll cells. Transforming growth factor 尾 (TGF 尾), fibroblast growth factor (bFGF), nicotinamide (Nicotinamide) and co-culture with islet cells were added to the cell culture medium to induce the differentiation of bone marrow stromal cells into pancreatic 尾 cells. The expression of insulin, somatostatin, glucagon and mRNA were detected by fluorescence immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). To determine whether bone marrow stromal cells differentiated into pancreatic 尾 cells. (2) Hoechst 33258 nuclear fluorescent dye was used to label bone marrow stromal stem cells. The labeled bone marrow stromal stem cells (BMSCs) were transplanted into the pancreatic tissues of rats. The pancreatic tissues of rats were taken every three days for frozen sections to observe the colonization of fluorescent labeled cells in the tissues. The expression of insulin in fluorescent cells was detected by fluorescence immunohistochemistry. (3) single intraperitoneal injection of streptozotocin (STZ) was used to induce diabetic rat model. Diabetic rats were randomly divided into two groups: bone marrow stromal cell transplantation group and medium injection group. Bone marrow stromal stem cells (BMSCs) were suspended in 200 渭 l serum-free medium and injected directly into pancreatic tissue by microinjector after laparotomy, and 200 渭 l serum-free medium was injected into pancreatic tissue in the medium injection group as control group. Blood glucose levels were measured at 3 days and 1 week after transplantation, and the changes of blood glucose in rats were observed. Results: (1) under the action of TGF 尾 cytokines, a few rat bone marrow stromal cells differentiated into insulin protein positive expression. Pancreatic 尾-like cells with positive expression of insulin and Pdx-1 gene, but negative expression of somatostatin and glucagon genes. Bone marrow mesenchymal stem cells co-cultured with islet cells, bone marrow stromal cells differentiated
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R329.2

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