再生胰腺提取物诱导人羊膜间充质干细胞定向分化为胰岛素分泌细胞

Differentiation of human amniotic mesenchymal stem cells into insulin-secreting cells induced by regenerating pancreatic extract

摘要:

利用天然生物诱导剂大鼠再生胰腺提取物 (Rgenerating pancreatic extract，RPE) 定向诱导人羊膜间充质干细胞 (Human amniotic mesenchymal stem cells，hAMSCs) 向胰岛素分泌细胞分化。切除大鼠60％胰腺刺激胰腺再生，而后制备RPE，以终浓度为20 mg/L的RPE诱导hAMSCs。实验通过形态学鉴定、双硫腙染色、免疫荧光分析、RT-PCR基因检测和高糖刺激胰岛素分泌等实验鉴定细胞诱导结果。实验结果显示P3代hAMSCs经RPE诱导后形态变化明显，诱导15 d后细胞呈簇状生长，经双硫腙染色可见棕红色细胞团；免疫荧光染色结果显示诱导细胞呈胰岛素阳性表达；RT-PCR实验证明诱导细胞阳性表达人胰岛相关基因Pdx1和insulin；高糖刺激实验证明培养液中有胰岛素成分产生，且分泌量随刺激时间的延长先增加而后趋于稳定。实验结果表明hAMSCs在体外经RPE诱导可以分化为胰岛素分泌细胞。

Abstract:

In this study, the natural biological inducer, rat regenerating pancreatic extract (RPE), was used to induce human amniotic mesenchymal stem cells (hAMSCs) into insulin-secreting cells. We excised 60% of rat pancreas in order to stimulate pancreatic regeneration. RPE was extracted and used to induce hAMSCs at a final concentration of 20 μg/mL. The experiment methods used were as follows: morphological-identification, dithizone staining, immumofluorescence analysis, reverse transcription-PCR (RT-PCR) and insulin secretion stimulated by high glucose. The results show that the cell morphology of passge3 hAMSCs changed significantly after the induction of RPE, resulting in cluster shape after induction for 15 days. Dithizone staining showed that there were scarlet cell masses in RPE-treated culture. Immumofluorescence analysis indicated that induced cells were insulin-positive expression. RT-PCR showed the positive expression of human islet-related genes Pdx1 and insulin in the induced cells. The result of insulin secretion stimulated by high glucose indicated that insulin increasingly secreted and then kept stable with prolongation of high glucose stimulation. In conclusion, hAMSCs had the potential to differentiate into insulin-secreting cells induced by RPE in vitro.