| 摘要: |
| 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、40 mmol/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。 |
| 关键词: 诱导多潜能干细胞 胰岛素分泌细胞 转录因子 细胞分化 |
| DOI： |
| 投稿时间：2013-01-20修订日期：2013-05-02 |
| 基金项目:国家自然科学基金(81101615, 81070656),中国博士后基金(2012M521107),江苏省自然科学基金 (BK2010276). |
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| Polygene-modified mouse induced differentiation of pluripotent stem cells into insulin-producing cells |
| FAN Xiang-jun 1,2,WANG Lei2 ,LU Yu-hua2 ,ZHU Ming-yan2 ,QIAN Hai-xin1*,WANG Zhi-wei2* |
(1. Department of General Surgery, the First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu, China
2. Department of General Surgery, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu, China
*Corresponding authors.) |
| Abstract: |
| 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-ef1a-Hygromicin-TRE-Oct4/Sox2/Klf4/cMyc) at a multiplicity of infection, and iPSCs were selected and identified. Then the iPSCs were infected with adenovirus (Ad-mPDX-1-IRES-GFP, Ad-mNeuroD1-IRES -GFP and Ad-mMafA-IRES -GFP) to induce differentiation into insulin-producing 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 Rex-1, and differentiate into three embryonic layers, which indicating that MEFs were successfully reprogrammed into iPSCs. Mouse iPSCs infected with Ad-PDX-1-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 cells. 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 into pancreatic islet B cells capable of insulin biosynthesis and secretion. |
| Key words: induced pluripotent stem cells insulin-secreting cells transcription factors cell differentiation |
