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目的观测胰岛素转录关键调控因子胰腺十二指肠同源框蛋白1(PDX-1)、神经分化因子1(NeuroD1)及肌腱膜纤维肉瘤癌基因同系物A(MafA)对小鼠诱导多潜能干细胞(iPS细胞)分化为胰岛素分泌细胞的作用。方法筛选鉴定小鼠胚胎成纤维细胞(MEFs)重编程的iPS细胞。以重组腺病毒Ad-mPDX-1-IRES-GFP、Ad-mNeuroD1-IRES-GFP、Ad-mMafA-IRES-GFP联合感染小鼠iPS细胞,体外培养后以RT-PCR检测胰岛B细胞功能基因表达;免疫荧光检测胰岛素蛋白表达及定位;ELISA检测不同糖浓度(0、5、10、20、30、40mmol/L)下胰岛素的分泌量。结果起源于MEFs的iPS细胞能形成边缘光整的致密克隆,表达干性基因Nanog、Rex-1、SSEA-1,并能在体内外分化为三胚层组织,显示MEFs被成功地重编程为iPS细胞。Ad-PDX-1-IRES-GFP、Ad-mNeuroD-IRES-GFP、Ad-mMafA-IRES-GFP感染的小鼠iPS细胞能分化为胰岛类B细胞,RT-PCR结果显示其胰岛B细胞功能基因的表达与小鼠胰岛B细胞株MIN6相似。免疫荧光检测可见胰岛类B细胞内有胰岛素表达。ELISA检测结果显示胰岛类B细胞对不同浓度葡萄糖有较好的反应性。结论胰岛素转录关键调控因子PDX-1、NeuroD1和MafA三者能协同作用,使小鼠iPS细胞分化为具有显著胰岛素合成和分泌能力的胰岛素分泌细胞。
Objective To observe the effects of pancreatic duodenal homeobox 1 (PDX-1), NeuroD1 and MafA on the induction of pluripotent stem cells (iPS cells) into insulin-secreting cells. Methods Screening identified iPS cells reprogrammed in mouse embryonic fibroblasts (MEFs). The mouse iPS cells were infected with the recombinant adenovirus Ad-mPDX-1-IRES-GFP, Ad-mNeuroD1-IRES-GFP and Ad-mMafA-IRES-GFP respectively and the expression of functional gene of islet B cells was detected by RT- ; Insulin protein expression and localization were detected by immunofluorescence; The amount of insulin secreted by different concentrations of glucose (0, 5, 10, 20, 30, 40mmol / L) Results The iPS cells derived from MEFs could form tight edge and compact clones expressing the dry genes Nanog, Rex-1 and SSEA-1 and differentiating into the three germ layers in vivo and in vitro, indicating that MEFs were successfully reprogrammed to iPS cell. The mouse iPS cells infected with Ad-PDX-1-IRES-GFP, Ad-mNeuroD-IRES-GFP and Ad-mMafA-IRES-GFP can differentiate into islet B cells. RT-PCR results showed that the islet B cell function gene The expression of mouse pancreatic islet B cell line MIN6 similar. Immunofluorescence showed that islet B cells had insulin expression. ELISA test results showed that islet B cells have better reactivity to different concentrations of glucose. Conclusion The key regulatory factors of insulin, PDX-1, NeuroD1 and MafA, can synergize with each other and differentiate the mouse iPS cells into insulin-secreting cells with significant insulin synthesis and secretion.