人脂肪间充质干细胞向胰岛素分泌细胞分化体系的建立和优化及miR-375过表达对分化的影响
发布时间：2018-08-22 09:05
【摘要】：糖尿病是一种常见的严重代谢性疾病,而胰岛移植是目前效果最好的治疗方法,但是胰岛来源匮乏严重制约了该疗法的使用。通过定向诱导或基因工程的方法可以使非胰β细胞具有胰岛素分泌功能,从而代替胰β细胞用于糖尿病的细胞治疗,为糖尿病患者带来了新希望。本研究以脂肪间充质干细胞为研究对象,建立了诱导其形成胰岛素分泌细胞的分化体系并研究了 miR-375过表达对分化的影响,为脂肪间充质干细胞在糖尿病治疗中的应用奠定了基础。基于胚胎干细胞分化为胰岛素分泌细胞(insulinproducing cells,IPCs)期间的关键步骤和规律,以及前期对人脂肪间充质干细胞(human adipose tissue-derived mesenchymal stem,haMSCs)胰向分化的探索,确立五种含有不 同诱导因子的分化方案。通过使用这五种方案分别对脂肪间充质干细胞进行诱导分化以及对分化细胞进行基因表达检测和细胞形态学比较,最终确定了以第一阶段添加小分子CYC、NOGGIN、B27和RA的基础高糖培养液;第二阶段添加Nic、GLP1、NOGGIN和B27的基础高糖培养液;第三阶段添加Nic、GLP1、IGF1和NOGGIN的IMDM培养液的三阶段分化方案为最优分化方案。我们通过使用该方案成功诱导haMSCs分化为IPCs,分化细胞表达胰腺相关基因Foxa2、Pdx1、Ngn3、Ins和Gcg,细胞胰岛素免疫荧光染色为阳性,分泌胰岛素的ELISA检测为阳性。并发现BMP信号通路对胰岛素分泌细胞的形成有调节作用,NOGGIN的加入有促进Ins表达的作用。实验室所保存的人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hucMSC)可以作为 haMSC 分化过程中同期处理的对照细胞。miR-375在胰腺、胰岛细胞发育和成熟起到重要的调控作用,控制其表达可能有助于干细胞向胰岛素分泌细胞分化。为了探究基于前期建立的诱导分化方案过表达miR-375后对分化究竟有何影响,我们利用慢病毒载体,成功构建稳定过表达miR-375的haMSC细胞株,并将其诱导分化为IPCs;同时,我们构建了一种在分化第三阶段瞬时过表达/抑制miR-375的脂质体转染体系,来探究过表达/抑制miR-375后对haMSC分化的影响。通过以上两种方法,得出:miR-375 和 Ins表达呈正相关;过表达 miR-375 可以降低分化所得细胞中 Gcg 的表达;在分化第三阶段起始瞬时过表达miR-375可以上调Ngn3;miR-375过表达对Pdx1、Foxa2基因表达影响不大。总之,过表达miR-375可以促进细胞向胰岛素分泌细胞分化,并减少向胰高血糖素分泌细胞分化。
[Abstract]:Diabetes mellitus is a common severe metabolic disease, and islet transplantation is the most effective treatment at present, but the lack of islet source seriously restricts the use of the therapy. By means of directed induction or genetic engineering, non-pancreatic 尾 cells can be secreted by insulin instead of pancreatic 尾 cells, which brings new hope to diabetic patients. In this study, adipose mesenchymal stem cells (FMSCs) were used to induce the differentiation of insulin-secreting cells and the effect of overexpression of miR-375 on differentiation was studied. It lays a foundation for the application of adipose mesenchymal stem cells in the treatment of diabetes. Based on the key steps and rules during the differentiation of embryonic stem cells into insulinproducing cells, and the preliminary exploration of pancreatic differentiation of human adipose mesenchymal stem cells (human adipose tissue-derived mesenchymal stemma haMSCs), five differentiation schemes containing different inducible factors were established. By using these five schemes to induce the differentiation of adipose mesenchymal stem cells and to detect the gene expression of the differentiated cells and compare the morphology of the cells, the basic high glucose medium was determined by adding small molecule CYCnGINB27 and RA in the first stage. In the second stage, the basic high glucose medium of Nickel GLP1NOGGIN and B27 was added, and in the third stage, the three-stage differentiation scheme of IMDM medium supplemented with Nickel GLP1IGF1 and NOGGIN was the best. We successfully induced the differentiation of haMSCs into IPCs by using this protocol. The differentiated cells expressed the pancreatic associated genes Foxa2, Pdx1, Ngn3, ins and Gcg. The cells were positive for insulin immunofluorescence staining and ELISA positive for insulin secretion. It was also found that BMP signaling pathway could regulate the formation of insulin-secreting cells. The addition of NOGGIN could promote the expression of Ins. Human umbilical cord mesenchymal stem cells (human umbilical cord mesenchymal stem cells hucMSC) can play an important role in the development and maturation of pancreatic islet cells. Controlling its expression may help stem cells differentiate into insulin-secreting cells. In order to investigate the effect of overexpression of miR-375 on differentiation based on the previously established induction protocol, we successfully constructed a stable haMSC cell line with overexpression of miR-375 by using lentivirus vector, and induced it to differentiate into IPCs. at the same time, We constructed a liposome transfection system for transient overexpression / inhibition of miR-375 in the third stage of differentiation to investigate the effect of overexpression / inhibition of miR-375 on the differentiation of haMSC. Through the above two methods, the positive correlation between the expression of miR-375 and Ins was found, and overexpression of miR-375 could decrease the expression of Gcg in differentiated cells. Transient overexpression of miR-375 in the third stage of differentiation could up-regulate the expression of Ngn3mmiR-375 and had little effect on the expression of Pdx1 + Foxa2 gene. In conclusion, overexpression of miR-375 can promote the differentiation of cells into insulin-secreting cells and reduce the differentiation to glucagon secreting cells.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1
本文编号：2196637
[Abstract]:Diabetes mellitus is a common severe metabolic disease, and islet transplantation is the most effective treatment at present, but the lack of islet source seriously restricts the use of the therapy. By means of directed induction or genetic engineering, non-pancreatic 尾 cells can be secreted by insulin instead of pancreatic 尾 cells, which brings new hope to diabetic patients. In this study, adipose mesenchymal stem cells (FMSCs) were used to induce the differentiation of insulin-secreting cells and the effect of overexpression of miR-375 on differentiation was studied. It lays a foundation for the application of adipose mesenchymal stem cells in the treatment of diabetes. Based on the key steps and rules during the differentiation of embryonic stem cells into insulinproducing cells, and the preliminary exploration of pancreatic differentiation of human adipose mesenchymal stem cells (human adipose tissue-derived mesenchymal stemma haMSCs), five differentiation schemes containing different inducible factors were established. By using these five schemes to induce the differentiation of adipose mesenchymal stem cells and to detect the gene expression of the differentiated cells and compare the morphology of the cells, the basic high glucose medium was determined by adding small molecule CYCnGINB27 and RA in the first stage. In the second stage, the basic high glucose medium of Nickel GLP1NOGGIN and B27 was added, and in the third stage, the three-stage differentiation scheme of IMDM medium supplemented with Nickel GLP1IGF1 and NOGGIN was the best. We successfully induced the differentiation of haMSCs into IPCs by using this protocol. The differentiated cells expressed the pancreatic associated genes Foxa2, Pdx1, Ngn3, ins and Gcg. The cells were positive for insulin immunofluorescence staining and ELISA positive for insulin secretion. It was also found that BMP signaling pathway could regulate the formation of insulin-secreting cells. The addition of NOGGIN could promote the expression of Ins. Human umbilical cord mesenchymal stem cells (human umbilical cord mesenchymal stem cells hucMSC) can play an important role in the development and maturation of pancreatic islet cells. Controlling its expression may help stem cells differentiate into insulin-secreting cells. In order to investigate the effect of overexpression of miR-375 on differentiation based on the previously established induction protocol, we successfully constructed a stable haMSC cell line with overexpression of miR-375 by using lentivirus vector, and induced it to differentiate into IPCs. at the same time, We constructed a liposome transfection system for transient overexpression / inhibition of miR-375 in the third stage of differentiation to investigate the effect of overexpression / inhibition of miR-375 on the differentiation of haMSC. Through the above two methods, the positive correlation between the expression of miR-375 and Ins was found, and overexpression of miR-375 could decrease the expression of Gcg in differentiated cells. Transient overexpression of miR-375 in the third stage of differentiation could up-regulate the expression of Ngn3mmiR-375 and had little effect on the expression of Pdx1 + Foxa2 gene. In conclusion, overexpression of miR-375 can promote the differentiation of cells into insulin-secreting cells and reduce the differentiation to glucagon secreting cells.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1
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