尼克酰胺与胰腺损伤提取物诱导小鼠骨髓间充质干细胞分化为胰岛素产生细胞的比较

Comparison between nicotinamide and rat pancreatic extract for inducing mouse mesenchymal stem cells to differentiate into insulin producing cells

目的探讨诱导小鼠骨髓间充质干细胞(BMSCs)分化为胰岛素产生细胞较好的方法。方法大鼠胰腺提取物和尼克酰胺分别诱导小鼠的BMSCs分化,经免疫组织化学、双硫腙染色、放射免疫学方法和RT-PCR技术(Ins-1、Ins-2、Glut-2、GK)鉴定两种诱导方法诱导细胞的分化程度。结果两种方法诱导后的细胞均可以表达胰岛素。双硫腙染色和胰岛素免疫组织化学的染色结果发现,两种分化后的细胞无明显差异。放射免疫学检测胰岛素及C肽片段:两种分化后的细胞表达的胰岛素随25mmol/L葡萄糖刺激时间的延长产生的胰岛素分泌曲线有明显差别,尼克酰胺分化的小鼠BMSCs产生的胰岛素产生细胞(IPCs)与正常小鼠胰岛细胞的曲线一致性欠佳,而大鼠胰腺损伤提取物分化的小鼠骨髓间充质干细胞产生的胰岛素产生细胞的分泌曲线一致性很好。尼克酰胺分化后的细胞不能产生C肽片段,而大鼠胰腺损伤提取物能够表达C肽片段。RT-PCR结果显示,尼克酰胺与大鼠胰腺提取物均可以表达胰岛细胞相关的几个mRNA(Ins-1、Ins-2、Glut-2、GK)。结论大鼠胰腺提取物与尼克酰胺相比,可能是一种较好的诱导BMSCs分化成熟产生胰岛素的诱导剂。

Objective By comparing rat pancreatic extract（RPE） with nicotinamide, we explored a more effective method for inducing mouse bone marrow mesenchymal stem cells（BMSCs） into insulin producing cells. Methods RPE and nicotinamide were used to trans-differentiate mouse BMSCs into insulin producing cells. After trans-differentiation, the two methods induced insulin producing cells （IPCs） were detected by immunocytochemistry to compare the insulin expression in cytoplasm and detected trans-differentiation extent by diphenylthiocarbazone（DTZ） staining. Insulin releasing curve of IPCs exposed to 25mmol/L glucose and C peptide expression were detected by Radioimmunoassay（RIA）. Four pancreatic islets associated mRNA （Ins-1 ,Ins-2,Glut-2 and GK） expression by RT-PCR technology. Then we analyzed and compared trans-differentiation effect between the two methods. Result From immunocytochemistry of insulin, we found that insulin producing cells trans-differentiated by both methods and all could secrete insulin and no significant staining extent difference was observed. And also there was no difference between them in DTZ stain and morphology pictures. However RIA showed the evident difference between them. The insulin releasing curve by the IPCs exposed to 25mmol/L glucose showed that nicotinamide induced IPCs were not like RPE induced cells which were more similar to mouse pancreatic islets. Moreover, the nicotinamide induced BMSCs could not express C peptide, and the RPE induced BMSCs expressed C peptide. RT-PCR showed nicotinamide and RPE induced BMSCs could all express the islets related mRNA （Ins-1, Ins-2, Glut-2, GK）. Conclusion As compared with Nicotinamide, RPE seems to be a more effective inducer to trans-differentiate BMSCs into IPCs.