摘要
背景:目前,体外已成功将骨髓间充质干细胞定向诱导分化为能够分泌胰岛素的胰岛样细胞团,但是对细胞的形态变化规律、胰岛样细胞团中内分泌细胞的类型及其相互关系的研究报道较少。目的:观察骨髓间充质干细胞定向分化为胰岛样结构过程中细胞的形态变化、内分泌细胞的组成及分布规律。方法:取生长良好的第3代骨髓间充质干细胞,扩增培养至细胞集落汇合占据瓶底面积80%,加入胰腺组织裂解液连续诱导培养。以双硫腙染色方法对骨髓间充质干细胞定向分化形成的细胞团进行初步筛选,Mallory染色鉴别胰岛内分泌细胞的类型及其分布;采用免疫荧光细胞化学染色法检测胰岛内分泌细胞中胰岛素、C肽、胰高血糖素和生长抑素蛋白表达。结果与结论:①双硫腙染色显示,随着诱导时间延长,阳性细胞数量逐渐增多;②Mallory染色显示,胰岛样细胞团内α样细胞位于周边,β样细胞分布在中央及周边,成纤维样细胞分布在周围;③免疫荧光染色显示,胰岛样细胞团内有胰岛素、C肽、胰高血糖素和生长抑素阳性细胞;④结果表明,在特定的微环境条件下,骨髓间充质干细胞能分化为胰岛样结构,内含α、β、δ样细胞,周围有类成纤维细胞包绕。
BACKGROUND: Bone marrow mesenchymal stem cells have been induced into islet-like cell mass in vitro. However, little is reported on the morphological changes of cells, the types of endocrine cells in the islet-like cell mass and their relationships.OBJECTIVE: To investigate the morphological changes of cells in the differentiation of bone marrow mesenchymal stem cells into islet-like structure and to explore the composition and distribution of endocrine cells.METHODS: Passage 3 bone marrow mesenchymal stem cells growing well were cultured and expanded until the cell colonies occupies 80% of the bottom of the culture bottle, and the pancreatic tissue lysate was added for continuous induction. Dithizone staining was used to screen the islet-like cell mass directly differentiated from bone marrow mesenchymal stem cells. The types and distribution of endocrine cells were identified by Mallory staining. Expressions of insulin, C-peptide, glucagon and somatostatin protein were detected by immunofluorescence cytochemical staining.RESULTS AND CONCLUSION:(1) Dithizone staining showed that the number of positive cells was increased over the induction time.(2)Mallory staining showed the red α-like cells were located in the periphery of the islet-like cell mass, the yellow β-like cells located in the center and periphery, and the light blue fibroblasts were distributed around the cell mass.(3) Immunofluorescence staining showed insulin, C-peptide,glucagon and somatostatin positive cells in the islet-like cell mass. To conclude, under certain microenvironment, bone marrow mesenchymal stem cells can differentiate into islet-like structures which containing α, β, δ-like cells, and are surrounded by fibroblast-like cells.
引文
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