小鼠骨髓间质干细胞转分化为胰岛样细胞的实验研究

发布时间：2018-04-02 11:39

本文选题：骨髓间质干细胞　切入点：胰岛　出处：《郑州大学》2006年硕士论文

【摘要】：目的：1型糖尿病是针对胰岛β细胞的特异性自身免疫性疾病，需终身依赖胰岛素治疗。但是胰岛素治疗并不能从根本上治愈该病，不仅给患者带来很大的痛苦，对社会和家庭也是沉重的负担。新的治疗策略就是寻找合适的细胞以替代β细胞发挥生理功能。然而，供体来源的匮乏以及移植免疫排斥反应极大地阻碍了该方法的推广和应用。骨髓间质干细胞(MSC)是一种具有自我更新和多向分化潜能，存在于骨髓的非造血成体干细胞，这类细胞呈现稳定的表型，易于分离、培养，，自我复制，有高度增殖潜力，而且在一定条件下具有跨系分化潜力。本研究采用于细胞培养及诱导分化技术，分离培养小鼠骨髓间质干细胞，使其转分化为分泌胰岛素细胞。材料与方法：实验用雄性成年昆明小鼠(4周～6周)，用5ml注射器冲出股骨、胫骨中的骨髓，分别培养于完全低糖DMEM培养基中1小时，含1％二甲基亚砜的无血清DMEM中3天和完全高糖DMEM中7天。取培养第1天、第3天、第10天的细胞置于倒置显微镜下观察细胞的生长情况及形态变化；培养第10天时细胞免疫化学染色检测细胞内胰岛素、胰高血糖素抗体；以RT-PCR法检测第1天和10天时细胞内Insulin和Glucagon基因的表达，用western blot检测第10天时细胞内胰岛素蛋白的表达，用双硫腙(DTZ)染色试验检测胰岛样细胞团内是否有Insulin合成，以及葡萄糖刺激的胰岛素释放试验检验胰岛样细胞是否能够初步模拟体内β细胞的生理功能。结果： 1．小鼠骨髓间质干细胞的原代培养接种后可见绝大部分细胞成圆球形，单个存在，偶可见未解离的3-5个细胞
[Abstract]:Objective Type 1 diabetes mellitus is a specific autoimmune disease for islet 尾 cells, which needs to be treated with insulin for life. But insulin therapy can not cure the disease fundamentally, and it not only brings great pain to the patients. It's also a heavy burden for society and families. The new treatment strategy is to find the right cells to replace beta cells. However, The lack of donor sources and allograft rejection have greatly hindered the development and application of this method. Bone marrow mesenchymal stem cells (MSCs) are non-hematopoietic adult stem cells with self-renewal and multi-differentiation potential. These cells exhibit stable phenotypes, easy to isolate, culture, self-replicate, have high proliferative potential, and have cross-line differentiation potential under certain conditions. Mouse bone marrow mesenchymal stem cells were isolated and cultured to differentiate into insulin-secreting cells. Materials and methods: male adult Kunming mice were used to culture bone marrow of femur and tibia with 5ml syringe for 1 hour, respectively. In serum free DMEM containing 1% dimethyl sulfoxide for 3 days and 7 days for complete high glucose DMEM, the cells were cultured on day 1, day 3 and day 10 to observe the growth and morphological changes of the cells under inverted microscope. The intracellular insulin and glucagon antibody were detected by immunocytochemical staining on the 10th day of culture, the expression of Insulin and Glucagon genes were detected by RT-PCR method on the 1st and 10th day, and the expression of insulin protein was detected by western blot on the 10th day. Dithizone dithizone (DTZ) staining was used to detect whether there was Insulin synthesis in islet like cells and glucose stimulated insulin release test was used to test whether islet like cells could mimic the physiological function of 尾 cells in vivo. Results:. 1. Primary culture of mouse bone marrow mesenchymal stem cells. After inoculation, most of the cells were spherical, single, and occasionally 3-5 cells were not dissociated.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329.2

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