人骨髓间充质干细胞早期和晚期间差异表达基因的鉴定

发布时间：2018-04-25 09:09

本文选题：人骨髓间充质干细胞 + 抑制消减杂交　；参考：《汕头大学》2008年硕士论文

【摘要】：间充质干细胞(Mesenchymal stem cells,MSCs)具有很强的自我更新能力,并具有很高的可塑性,在组织工程及临床细胞治疗等方面具有广阔的应用前景,所以间充质干细胞已成为干细胞研究的热点,如何在体外培养过程中维持MSCs未分化状态已成为其中的核心问题之一。而到目前为止对调节MSCs生长和未分化状态的分子机理方面的研究甚少。因此为了进一步了解维持MSCs未分化状态的机制,本论文采用自行建立培养的人骨髓间充质干细胞(human bone marrow mesenchymal stem cells, hBM-MSCs)克隆,应用抑制消减杂交(suppression subtractive hybridization, SSH)结合半定量RT-PCR技术筛选和鉴定hBM-MSCs早期和晚期间差异表达的基因。本研究首先分离培养hBM-MSCs,再分别选取传代培养至第五代和第二十代的细胞作为实验方(tester)和驱动方(driver),进行抑制消减杂交,构建cDNA消减杂交文库。然后将消减杂交后的PCR产物克隆到pGEM-T easy载体中,随后挑取阳性克隆进行测序,并与GenBank人基因库中已公布的核酸序列进行同源性比较分析。结果表明挑取的117个克隆分别代表21个已知的人基因,同源性都达到90%以上,这些基因分别具有不同的生物学功能。为进一步确定消减杂交的结果,进行半定量RT-PCR分析,结果发现21个基因中有6个基因在早期hBM-MSCs中的表达量都是晚期中表达量的2倍以上,它们分别是rab23,ddx21 ,tnfaip3 ,nomo2 ,pabpc3及cnn3,这些基因可能对维持hBM-MSCs的未分化状态和调节早期hBM-MSCs的生长具有重要作用。本研究将为这些基因的进一步功能研究以及阐明维持hBM-MSCs未分化状态的机制打下基础,而且有助于改变和控制hBM-MSCs的生长和分化的研究,进一步推动hBM-MSCs在组织工程上的应用。
[Abstract]:Mesenchymal stem cells (MSCs) have strong self-renewal ability and high plasticity, and have broad application prospects in tissue engineering and clinical cell therapy, so mesenchymal stem cells have become a hot spot in stem cell research. How to maintain the undifferentiated state of MSCs in vitro has become one of the core problems. So far, little has been done on the molecular mechanism of regulating the growth and undifferentiated state of MSCs. Therefore, in order to further understand the mechanism of maintaining the undifferentiated state of MSCs, human bone marrow mesenchymal stem cells (hBM-MSCs) clones were isolated from human bone marrow mesenchymal stem cells (BMSCs). Suppression subtractive hybridization (SSH) combined with semi-quantitative RT-PCR technique was used to screen and identify differentially expressed genes between early and late hBM-MSCs by suppression subtractive hybridization (SSH). In this study, hBM-MSCs were isolated and cultured, and then the subtractive hybridization (cDNA) library was constructed by subtractive hybridization of cells from the fifth generation and the 20th generation. Then the subtractive PCR products were cloned into the pGEM-T easy vector, then the positive clones were sequenced and compared with the nucleic acid sequences published in the GenBank human gene bank. The results showed that 117 clones represented 21 known human genes with more than 90% homology, and these genes had different biological functions. In order to further determine the results of subtractive hybridization, semi-quantitative RT-PCR analysis showed that 6 of 21 genes expressed more than twice as much in early hBM-MSCs as in late stage. These genes are rab23ddx21 tnfaip3 / nfaip2 / pabpc3 and cnn3 respectively. These genes may play an important role in maintaining the undifferentiated state of hBM-MSCs and regulating the growth of early hBM-MSCs. This study will lay a foundation for the further study of the function of these genes and clarify the mechanism of maintaining the undifferentiated state of hBM-MSCs. It will also be helpful to change and control the growth and differentiation of hBM-MSCs and further promote the application of hBM-MSCs in tissue engineering.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329;R346

【相似文献】