体外定向诱导人脐带间充质干细胞分化为胰岛素分泌细胞的研究

Inducing human umbilical cord Wharton's jelly-derived mesenchymal stem cells(HuMSCs) into insulin secreting cells in vitro

目的研究人脐带间充质干细胞(human umbilical cord Wharton s jelly-derived mesenchymal stem cells,HuMSCs)向胰岛素分泌细胞分化的潜能,为胰岛细胞移植治疗Ⅰ型糖尿病提供新的细胞来源。方法体外分离培养HuMSCs,在胰岛细胞培养条件下经药物(尼克酰胺、β-巯基乙醇和高糖)定向诱导其分化;倒置相差显微镜下观察诱导后细胞形态;免疫组化、RT-PCR对诱导细胞进行胰岛β细胞标记鉴定;双硫腙染色鉴定锌离子表达;放射免疫法检测胰岛素含量。结果诱导后HuMSCs由长梭形逐渐变为多角形、类圆形,部分聚集成团;免疫细胞化学染色显示,HuMSCs经诱导后表达人胰岛素和胰高血糖素抗原;RT-PCR检测显示诱导后HuMSCs表达了人胰岛素基因和PDX-1基因;双硫腙染色呈棕红色。结论HuMSCs在体外胰岛细胞培养条件诱导下,具有向胰岛素分泌细胞分化的潜能,这种细胞可能为Ⅰ型糖尿病提供一条新的治疗途径。

Objective To explore the possibility of inducing the differentiation of human umbilical cord Wharton＇s jelly-derived mesenchymal stem cells （HuMSCs） into insulin secreting cells in vitro. Methods We isolated and cultured HuMSCs, and induced HuMSCs to differentiate into insulin secreting cells in the islet cell culture condition. The morphology of HuMSCs after induction was monitored by inversion phase contrast microscope. The insulin and glucagon protein were detected by immunocytochemical methods, and Human insulin gene and PDX-1 gene were detected by reverse transcription- polymerase chain reaction （RT-PCR）. Zinc hydronium was detected by Dithizon staining and insulin level was detected by radioimmunoassay . Results Under induction, the morphology of HuMSCs gradually changed from fihroblast to round shape and some of them had the trend of forming clusters. Immunceytochemical examination showed that the expression of human insulin and glucagon was positive after treatment with nicotinic amide and β-mercaptoethanol. HuMSCs induced by medicine can express insulin and PDX-Ⅰ gene by RT-PCR. Dithizon stain showed that the cytoplasm of HuMSCs was stained in brownish red color after induction. The results of radi y showed that the medicine induced HuMSCs secreted significant amount of insulin when compared with that of the control group （ P 〈 0. 05）. Conclusion HuMSCs can be differentiated into insulin secreting cells in vitro. HuMSCs have the potential to become an excellent candidate in β cell replacement therapy of diabetes.