体外分步诱导胰腺导管干细胞分化为胰岛β细胞

A step-by-step method for differentiating pancreatic duct stem cells into islets β cells in vitro

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 method for differentiating pancreatic duct stem cells into islets β cells in vitro. Methods The pancreatic stern cells were isolated and purified from SD rats, which were then cultured and expanded in vitro. Pancreatic duct stem cells were differentiated using a four-step method. The new produced islet-like clusters （ILCs） were harvested and examined by immunofluorescence stain and RT--PCR. The ability of insulin secretion was evaluated through glucose stimulated insulin secretion （GSIS）. Results The pancreatic duct cells isolated and purified had the character of stem cells. These stern cells could transform to ILCs which had spherical structure after cultured and differentiated by a four-step method. The results of immunofluorescence stain revealed that insulin and glucagon stain of the ILCs were positive. The results of RT-PCR revealed that the ILCs also had the expression of insulin and glucagon. The secretion amount of insulin of the ILCs was （1.1 ± 0. 2） ng/ml and （2.7 ± 0.2） ng/ml at low and high concentration of glucose, respectively, and the stimulation index was 2. 52. Conclusion The new differentiation and induction system can obtain ILCs that possess spherical structure and insulin secretion ability, which may provide a more effective method to obtain new sources of βcells for islet transplantation.