体外构建大鼠PDX-1基因克隆载体及体外诱导人脐带间充质干细胞向胰岛素分泌细胞分化

发布时间：2018-03-23 08:42

本文选题：PDX-1　切入点：cDNA　出处：《安徽医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：构建PDX-1基因的克隆载体，为真核表达载体的构建及研究PDX-1诱导干细胞分化的作用机制提供基础。方法：采用Trizol方法从大鼠胰岛细胞瘤细胞株中提取RNA，通过RT-PCR方法在体外扩增大鼠PDX-1cDNA，琼脂糖凝胶电泳鉴定，割胶纯化后回收目的基因，与pMD-18T克隆载体连接构建，，经限制性内切酶双酶切及DNA序列分析鉴定pMD-18T-PDX-1的正确构建。结果：正确构建了含有PDX-1cDNA的克隆载体pMD-18T-PDX-1，其中PDX-1cDNA全长为908bp。结论：在体外成功克隆了PDX-1基因，并正确构建了PDX-1基因克隆载体，对进一步进行PDX-1在真核细胞中的表达及研究PDX-1在干细胞定向分化中的作用具有重要的意义。目的：体外培养人脐带间充质干细胞，将间充质干细胞诱导分化为胰岛素分泌细胞，探讨诱导分化过程中干细胞向胰岛素分泌细胞分化的潜能及功能变化趋势。方法：体外培养人脐带间充质干细胞，培养至贴壁80%，将间充质干细胞分两组，A组为诱导组，B组为对照组。A组先后予以1mmol/L2-巯基乙醇培养2天，10ng/ml表皮生长因子、10ng/ml碱性成纤维细胞生长因子及2%B27培养7天，之后添加20mmol/L尼克酰胺及艾塞那肽培养7天。B组不添加试剂，继续换液培养。通过观察细胞形态变化、RT-PCR体外扩增两组细胞的PDX-1基因的cDNA、放射免疫法测定两组细胞胰岛素分泌量三种方法来鉴定胰岛素分泌细胞。结果：（1）通过形态学观察，可见未分化的脐带间充质干细胞呈长梭形，分界清楚，传代后细胞呈纤维样生长，而A组细胞逐渐变小，呈圆形，折光性变强，细胞聚集成团，类似胰岛样细胞，B组细胞仍呈长梭形，分界清楚，纤维样生长；（2）RT-PCR： A组细胞可扩增出PDX-1基因的cDNA，经琼脂糖凝胶电泳鉴定，PDX-1目的片段为912bp，与pubmed公布基因片段符合；B组细胞不能扩增出人PDX-1基因的cDNA。（3）通过放射免疫法测定两组细胞胰岛素分泌量，A组可检测出胰岛素分泌，B组未检出胰岛素分泌。结论：成功在体外微环境下将人脐带间充质干细胞诱导分化为胰岛素分泌细胞，为干细胞在糖尿病治疗领域的应用提供基础。
[Abstract]:Aim: to construct the clone vector of PDX-1 gene for the construction of eukaryotic expression vector and to study the mechanism of differentiation of stem cells induced by PDX-1. Methods: Trizol method was used to extract RNAs from rat islet cell tumor cell lines. Rat PDX-1cDNAwas amplified by RT-PCR method in vitro, and identified by agarose gel electrophoresis. The target gene was purified by tapping and constructed by ligation with pMD-18T clone vector. The correct construction of pMD-18T-PDX-1 was identified by restriction endonuclease digestion and DNA sequence analysis. Results: the clone vector pMD-18T-PDX-1 containing PDX-1cDNA was constructed correctly, in which the total length of PDX-1cDNA was 908bp. Conclusion: PDX-1 gene was successfully cloned in vitro and PDX-1 gene clone vector was constructed correctly, which is of great significance for further expression of PDX-1 in eukaryotic cells and study of the role of PDX-1 in stem cell differentiation. Aim: to culture human umbilical cord mesenchymal stem cells and differentiate them into insulin secreting cells. Methods: human umbilical cord mesenchymal stem cells were cultured in vitro. The mesenchymal stem cells were divided into two groups: group A: induction group: group B: control group. Group A was treated with 1 mmol / L 2-mercaptoethanol for 2 days and then cultured for 2 days with 10 ng / ml epidermal growth factor 10 ng / ml basic fibroblast growth factor and 27 days for 7 days. Group B was treated with 20mmol/L nicotinamide and Isenapeptide for 7 days. The PDX-1 gene cDNAs of the two groups of cells were amplified by RT-PCR in vitro and the insulin secretion of the two groups was determined by radioimmunoassay to identify the insulin-secreting cells. Results from morphological observation, the undifferentiated umbilical cord mesenchymal stem cells were shown to be fusiform, with clear boundary and fibroid growth after passage, while the cells in group A gradually became smaller, round, refractive change, and the cells gathered into clusters. The cells in group B of islet like cells were still fusiform, and the boundaries were clear. The cDNAs of PDX-1 gene were amplified by agarose gel electrophoresis (agarose gel electrophoresis). The target fragment of PDX-1 was 912bp.The cDNA of PDX-1 gene could not be amplified from pubmed group B cells by radioimmunoassay (RIA). Insulin secretion was detected in group A and no insulin secretion was detected in group B. Conclusion: human umbilical cord mesenchymal stem cells were successfully induced to differentiate into insulin secreting cells in vitro, which provided the basis for the application of stem cells in the field of diabetes treatment.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329;R587.1

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