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目的应用INGAP多肽在体外建立新的分步诱导分化体系诱导胰岛新生。方法分离纯化SD大鼠的胰腺导管干细胞。并体外扩增培养,取2～6代细胞进行分步诱导分化,于分化末期添加INGAP多肽,待诱导结束后收获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的胰岛素分泌能力。结果 (1)分离纯化的胰腺导管干细胞经过四步诱导后最终可形成立体结构的ILCs,其insulin和glucagon染色均为阳性,RT-PCR检测该ILCs也有insulin和glucagon基因的表达。(2)GSIS结果:新生ILCs和新分离胰岛的高糖组胰岛素释放量均显著高于低糖组(P<0.01);新生ILCs的胰岛素刺激指数接近新分离胰岛(P>0.01)。结论通过建立新的包含INGAP多肽的体外分步诱导分化体系,能够获得具有立体结构和一定胰岛素分泌能力的立体ILCs,为获取胰岛移植所需的β细胞提供了更有效的方法。
Objective To establish a new step-by-step induction of differentiation system using INGAP peptides to induce islet neovascularization. Methods Pancreatic ductal stem cells were isolated and purified from SD rats. And cultured in vitro. Cells of passage 2 to passage 6 were induced to differentiate in stages, and INGAP polypeptide was added at the end of differentiation. Neonatal islet-like cell mass (ILCs) were harvested after induction. Immunofluorescence and RT-PCR were performed respectively Insulin secretion of ILCs was assessed by a glucose-stimulated insulin release assay (GSIS). Results (1) The isolated and purified pancreatic ductal stem cells could finally form stereostructured ILCs after four-step induction. The positive cells were positive for insulin and glucagon, and the expression of insulin and glucagon gene was also detected by RT-PCR. (2) GSIS results: The insulin release of high glucose group was significantly higher than that of low glucose group (P <0.01). The insulin stimulating index of newborn ILCs was close to newly isolated islets (P> 0.01). Conclusion The establishment of a novel INGAP-containing in vitro differentiation system can provide stereostructural and insulin-secreting stereo ILCs, which provides a more effective method for obtaining β cells for islet transplantation.