诱导人脂肪干细胞分化为胰岛素分泌细胞:三种不同诱导分化方案的比较

Generating insulin-producing cells from human adipose-derived stem cells: Comparison of three differentiation protocols

目的将脂肪干细胞诱导分化为胰岛素分泌细胞是糖尿病治疗的新策略。目前已报道的诱导分化方案效率差异较大。本研究拟对三种诱导分化方案的分化效率进行比较。方法将第三代脂肪干细胞自聚集成微球,使用三种诱导分化方案(一步诱导法、两步诱导法、三步诱导法)。采用PCR和免疫荧光检测三种方案中关键基因和蛋白的表达水平,通过葡萄糖刺激胰岛素分泌实验检测终产物的功能。结果对比三种方案,三步诱导法诱导出的胰岛素分泌细胞其关键转录因子INS、NKX6.1、PDX1的基因表达量明显增加。关键蛋白C肽和NKX6.1/PDX1在胰岛素分泌细胞中共表达。三步诱导法能显著提高产物的胰岛素分泌量。结论在相同条件下,三步诱导法的分化效率优于其他两种方案。本研究为建立高效、安全的胰岛素分泌细胞诱导分化方案提供了依据。

Objective The induced differentiation of adipose stem cells into insulin-secreting cells is a new strategy for the treatment of diabetes mellitus.The efficiency of the induced differentiation schemes reported so far varies greatly.This study aims to compare the differentiation efficiency of three induced differentiation schemes.Methods Fat samples from the same batch were collected to extract fat stem cells for passage culture,and the third-generation fat stem cells were self-aggregated into balls for induction and differentiation in different phases(one-step method,two-step method and three-step method).The expression levels of key genes and proteins in the products of the three schemes at the corresponding stages were detected by PCR and immunofluorescence,and the function of insulin secreting cells was detected by glucose stimulation of insulin secretion experiment.Results Compared with other schemes,the gene expressions of the key transcription factors INS,NKX6.1 and PDX1 in the insulin secreting cells induced by the three-step method were significantly increased,and there were significant differences.Key protein C-peptide and NKX6.1/PDX1 were co-expressed in insulin-secreting cells.Functionally,the insulin secreting cells induced by the three-step method can significantly increase insulin secretion under high glucose stimulation.Conclusion Under the same differentiation conditions,the differentiation efficiency of adipose stem cells induced into insulin secreting cells by the three-step method is superior to that by the two-step method and the one-step method,which provides a basis for establishing an efficient and safe induction differentiation scheme of insulin secreting cells.