应用INGAP多肽体外分步诱导胰岛新生研究

The research on inducing islet neogenesis using INGAP-related pentadecapeptide in vitro

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 method for inducing islet regeneration with islet neogenesis associated protein （INGAP） related pentadecapeptide （INGAP-PP） in vitro. Methods The pancreatic duct stem cells were isolated and purifed from SD rats, then these ceils were cultured and expanded in vitro. For differentiation, cells from pasages 2-6 were allowed to differentiate with step by step mothod. At the last stage of the differentiation, the cells were cultured in the medium supplemented with INGAP-PP. The newly produced islet-like clusters （ILCs） were harvasted and detected by immunofluorescence stain and RT-PCR. Meanwhile, the ability of insulin secretion was evaluated through glucose stimulated insulin secretion （GSIS）. Results The isolated and purified pancreatic duct stem cells could transform to ILCs which had spherical structure after cultured and differentiated with INGAPPP 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 demonstrated that the ILCs also had the expression of insulin and glucagon. The results of GSIS showed that the amount of released insulin was significantly larger in high glucose group than in low glucose group in both new ILCs and new isolated islets （1.1 ±0. 2 ng/ml vs 2.7±0.2 ng/ml, 6.9±0.3 ng/ml vs 21.4±2.7ng/ml, respectively, allP〈0.01）; the stimulation index（SI）of new ILCs and new isolated islets was in no significant difference （2.6±0. 4 and 3.1±0. 4, respectively, P〈0.01）. Conclusions With INGAP-PP in vitro, we can obtain ILCs that possess spherical structure and insulin secretion ability. This study provides a more effective method to obtain new sources of β cells for islet transplantation.