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目的建立新的体外胰腺导管干细胞分化为胰岛β细胞的分步诱导分化体系。方法分离纯化SD大鼠的胰腺导管干细胞,体外扩增后进行分步诱导分化,收获的新生类胰岛样细胞团(ILCs)分别行免疫荧光和RT-PCR检测,并通过葡萄糖刺激的胰岛素释放试验(GSIS)评价ILCs的体外功能。结果分离纯化的胰腺导管干细胞经培养及四步诱导分化后最终可形成ILCs。免疫荧光结果显示该ILCs胰岛素和胰高血糖素染色均为阳性,RT-PCR检测也有胰岛素和胰高血糖素基因的表达。新生ILCs在低糖和高糖刺激下的胰岛素释放量分别为(1.1±0.2)ng/ml和(2.7±0.2)ng/ml,刺激指数为2.52倍。结论通过建立新的体外胰腺导管干细胞分化为胰岛β细胞的分步诱导分化体系,获得具有立体结构且有一定胰岛素分泌能力的ILCs,为获得胰岛移植的β细胞来源提供了更有效的途径。
Objective To establish a new step-by-step differentiation system of pancreatic ductal stem cells into pancreatic islet β cells in vitro. Methods Pancreatic ductal stem cells were isolated and purified from SD rats. Differentiation and differentiation were performed in vitro. Neonatal islet cell clusters (ILCs) harvested were detected by immunofluorescence and RT-PCR, respectively. The glucose-stimulated insulin release assay (GSIS) to evaluate the in vitro function of ILCs. Results The isolated and purified pancreatic ductal stem cells could finally form ILCs after being cultured and differentiated in four steps. Immunofluorescence results showed that the ILCs were positive for both insulin and glucagon staining and that the expression of insulin and glucagon genes was also detected by RT-PCR. The insulin release of neonatal ILCs was (1.1 ± 0.2) ng / ml and (2.7 ± 0.2) ng / ml, respectively, with a stimulus index of 2.52-fold under low and high glucose stimulation. Conclusion The establishment of a new step-by-step differentiation system of pancreatic ductal stem cells into islet β cells in vitro can provide ISTs with stereostructure and certain insulin secretion capacity, which provides a more effective way to obtain the source of β cells for islet transplantation.