多基因修饰小鼠诱导多潜能干细胞分化为胰岛素分泌细胞

Polygene-modified mouse induced differentiation of pluripotent stem cells into insulin-producing cells

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 evaluate the effects of key insulin gene transcription regulators （PDX-1, NeuroD1 and MafA） on the differentiation of induced pluripotent stem cells （iPSCs） into insulin-producing cells. Methods Mouse embryonic fibroblasts （MEFs） were infected with lentivirus （LV-efla-Hygromicin-TRE-Oct4/Soz2/Klf4/cMyc） at a multiplicity of infection, and iPSCs were seIected and identified. Then the iPSCs were infected with adenovirus （Ad-mPDX-I-IRES-GFP, Ad- mNeuroDl-IRES -GFP and Ad-mMafA IRES -GFP） to induce differentiation into insuliwproducing cells in vitro. RT-PCR was applied to detect expression of functional genes in pancreatic islet B cells; immunofluorescence was used to examine the expression and location of insulin protein; and ELISA was used to determine the volumes of secreted insulin at different concentrations of glucose （0,5,10,20,30, and 40 mmol/L）. Results The iPSCs derived from MEFs could form intensive clones with smooth boundary, express embryonic stem cell-specific cell surface markers, including Nanog, SSEA-1 and Rea~ 1, and differentiate into three embryonic layers, which indicating that MEFs were successfully reprogrammed into iPSCs. Mouse iPSCs infected with Ad-PDX-I-IRES-GFP, Ad-mNeuroD-IRES-GFP, and Ad-mMafA-IRES-GFP could differentiate into pancreatic islet B cells. RT-PCR results showed that polygene-modified iPSCs and pancreatic islet B-cell line MIN6 had similar gene expression profile. Immunofluorescence analyses confirmed insulin expression in the differentiated ceils. Results of ELISA showed that polygene-modified iPSCs had a satisfactory response to different concentrations of glucose. Conclusion Key insulin gene transcription regulators PDX-1, NeuroD1 and MafA can work synergistically to induce mouse iPSCs differentiation intopancreatic islet B cells capable of insulin biosynthesis and secretion.