骨髓间充质干细胞体外诱导分化为胰岛样细胞的研究

发布时间：2018-10-23 13:11

【摘要】： 本实验探讨了骨髓间充质干细胞(MSCs)体外原代的培养方法,及MSCs传代后在某些诱导条件下,分化为胰岛样细胞的实验方法。通过免疫细胞化学实验及动物实验,验证了MSCs在体外成功诱导分化为胰岛样细胞,并且可以修复坏死胰腺细胞。在MSCs的原代培养过程中,采用高速离心法及贴壁法分离纯化细胞。选用含10%胎牛血清(FBS)的低糖DMEM培养基作为扩增细胞的培养基。选择第三代状态良好的MSCs,进行诱导实验。采用三种不同的诱导途径,对诱导过程进行比较。发现最佳诱导方法如下:向生长状态稳定的第三代MSCs内加入内含碱性成纤维细胞生长因子(bFGF)(10ng/ml),表皮细胞生长因子(EGF)(10ng/ml)的无血清的LG-DMEM培养基,培养6～7d;然后,换用无血清的高糖DMEM培养基,添加胰腺条件培养液、胰岛素(25mg/ml)、地塞米松(10μg/l)、尼克酰胺(10mM/l),继续培养6～7d;在显微镜下观察MSCs,可以看到细胞形态渐发生改变,细胞由原来的梭形渐变为圆形及不规则形,最后细胞聚集成团、悬浮逐渐增多。经过细胞鉴定实验,证明已成功诱导分化为胰岛样细胞。这一研究,为临床胰腺类疾病(如胰腺炎及糖尿病等)的治疗提供了一个新的方向。
[Abstract]:The primary culture of bone marrow mesenchymal stem cells (MSCs) in vitro and the differentiation of MSCs into islet like cells were studied. The results of immunocytochemistry and animal experiments showed that MSCs could be successfully induced to differentiate into islet like cells in vitro and could repair necrotic pancreatic cells. During the primary culture of MSCs, the cells were isolated and purified by high speed centrifugation and adherent method. The low sugar DMEM medium containing 10% fetal bovine serum (FBS) was used as the medium for cell expansion. The third generation MSCs, with good state was selected for induction experiment. Three different ways of induction were used to compare the induction process. The results showed that the optimal induction methods were as follows: adding serum-free LG-DMEM medium containing basic fibroblast growth factor (bFGF) (10ng/ml) and epidermal growth factor (EGF) (10ng/ml) into the third generation MSCs of stable growth state, and culturing for 6 to 7 days. In addition to serum-free high-glucose DMEM medium, supplemented with pancreatic conditioned medium, insulin (25mg/ml), dexamethasone (10 渭 g / 1), and nicotinamide (10mM/l), the cells were cultured for 6 to 7 days. The cells gradually changed from fusiform to round and irregular. Finally, the cells gathered into clusters and gradually increased. The differentiation into islet-like cells was successfully induced by cell identification. This study provides a new direction for the treatment of pancreatic diseases such as pancreatitis and diabetes.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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