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目的体外诱导大鼠骨髓间质干细胞(BMSCs)分化为胰岛样细胞。方法采用分步法体外诱导后,间接免疫荧光法鉴定诱导前后细胞nestin、胰岛素蛋白表达;RT-PCR法检测诱导前后胰岛转录因子mRNA表达;EHSA检测诱导后细胞葡萄糖刺激的胰岛素分泌。结果免疫荧光染色显示诱导5 h,nestin阳性细胞为(44.6±7.3)%。诱导24 h,nestin阳性细胞增至(61.8±8.4)%。此后,nestin阳性细胞数目开始下降,诱导第14天后,nestin表达基本消失;同时诱导后的细胞可以表达胰岛素蛋白。另一方面,RT-PCR结果显示诱导后细胞可以表达胰岛素-1、葡萄糖转运子-2及其转录因子mRNA。ELISA结果显示不同浓度的葡萄糖刺激的胰岛素分泌量不同,5 mmol/L和25mmol/L葡萄糖刺激的胰岛素分泌量分别为(25.53±6.49)和(53.26±7.56)mU/L,而诱导前MSCs不具备上述特点。结论大鼠BMSCs体外可以诱导成为胰岛素分泌细胞。
Objective To induce differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into islet-like cells in vitro. Methods After in vitro induction with a stepwise method, indirect immunofluorescence was used to identify the expression of nestin and insulin protein before and after induction. RT-PCR was used to detect the mRNA expression of islet transcription factor before and after induction, and EHSA was used to detect glucose-induced insulin secretion after induction of EHSA. Results Immunofluorescence staining showed that nestin-positive cells were (44.6±7.3)% at 5 h of induction. At 24 hours of induction, nestin-positive cells increased to (61.8±8.4)%. Since then, the number of nestin-positive cells began to decline, and after 14 days of induction, the expression of nestin almost disappeared; meanwhile, the induced cells could express insulin protein. On the other hand, RT-PCR results showed that after induction, cells can express insulin-1, glucose transporter-2, and transcription factor mRNA. ELISA results showed that different doses of glucose stimulated insulin secretion differently, 5 mmol/L and 25 mmol/L glucose-stimulated insulin secretion were (25.53±6.49) and (53.26±7.56) mU/L, respectively, and MSCs were not pre-induction With the above characteristics. Conclusion Rat BMSCs can be induced to become insulin secreting cells in vitro.