【摘要】 目的:探讨人工模拟胰腺微环境诱导下骨髓间充质干细胞是否可以转分化为胰岛素产生细胞,并评价其功能。方法:采集兔骨髓间充质干细胞,分为2组。实验组将胰腺碎片整合到镍包被的微载体中,重建胰腺微环境,进行细胞培养,对照组为同期培养的未经诱导的细胞。骨髓间充质干细胞在微环境中培养3～4周后,观察是否有胰岛样细胞团形成。ELISA法检测细胞悬液中胰岛素含量,荧光免疫组化检测细胞团胰岛素和C-肽蛋白的表达,荧光原位杂交(FISH)检测胰岛素和C-肽mRNA的表达,流式细胞仪检测分化的细胞在高糖环境下继续培养3周是否发生凋亡。结果:培养3～4周后倒置显微镜下观察到胰岛样细胞团。实验组基础相胰岛素分泌量为(308.50±14.54)mIU/L,在高糖刺激下为(414.30±28.94)mIU/L,2者相比,差异有统计学意义(t=5.68,P<0.01);高糖刺激下实验组的胰岛素分泌量高于对照组的(5.21±1.04)mIU/L(t=34.60,P<0.01)。分化的胰岛样细胞团表达C-肽和胰岛素mRNA与蛋白。分化的细胞在高糖环境下继续培养3周未发生凋亡。结论:人工模拟胰腺微环境可以诱导骨髓间充质干细胞转分化为有功能的胰岛素产生细胞,有望替代胰岛细胞治疗糖尿病。更多还原

【Abstract】 Aim:To observe whether mesenchymal stem cells(MSCs) could trans-differentiate to insulin-producing cells under manipulated microenvironment and assess their function.Methods:We delivered pancreatic pieces into Niobium-Coated Dynamatrix to construct a simulated pancreatic microenvironment.MSCs cultured upon simulated pancreatic microenvironment were selected as experimental group. MSCs cultured without induction at the same period were selected as control group.Bone marrow derived MSCs were cultured upon the microenvironment for 3 to 4 weeks.Differentiated islet-like cells were observed. Insulin in supernatant was measured by ELISA. Insulin and c-peptide expression was verified by fluorescent immunocytochemistry and fluorescence in situ hybridization (FISH). Apoptosis of islet-like masses in high glucose DMEM was detected by flow cytometry.Results: After 3 to 4 weeks culture,typical islet-like masses were observed. Insulin secreted by differentiated cells with and without glucose stimulation were (414.30±28.94) and (308.50±14.54) mIU/L (t=5.68,P<0.01).The insulin secreted by differentiated cells with glucose stimulation was much higher than that of undifferentiated cells [(5.21±1.04) mIU/L,t=34.60,P<0.01)].Insulin and c-peptide expression were positive both in protein and mRNA levels. The trans-differentiated islet-like mass did not show any indication of apoptosis in high glucose DMEM for another 3 weeks culture.Conclusion: The simulated microenvironment could induce MSCs to differentiate into functional islet-like cells,which is hopeful to replace β-cell in treatment of diabetes.更多还原