人骨髓间充质干细胞早期和晚期间差异表达基因的鉴定
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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。
Mesenchymal stem cells (MSCs) can self-renew, are multipotent cells and can differentiate into many different cells. They are important cell souece for tissue engineering and cell therapy. So far, little is known about how MSC growth and differentiation is regulated, and this study tried to gain some insight into this question by identification of genes differentially expressed in human bone marrow mesenchymal stem cells (hBM-MSCs) between early passages and late passages. hBM-MSCs at passage 5 and passage 20 were used as a tester and a driver respetively for subtractive hybridization experiment. PCR product derived from subtractive hybridization was cloned into pGEM-Teasy vector. A total of 117 positive clones were taken for DNA sequencing. DNA sequence of each clone was compared with GenBank database for gene homologes, and 21 different human known genes were identified representing the 117 clones. DNA sequence homologies between each of these 21 genes and their respective known genes were greater than 90%. To confirm the differential expression of these genes, semi-quantitative RT-PCR analysis were performed, and it showed that 6 genes were indeed differentially expressed in hBM-MSCs between early passages and late passages by more than 1 fold. These genes included rab23, ddx21, tnfaip3, nomo2, pabpc3 and cnn3. These genes may play an important role in maitaning and regulating hBM-MSCs growth and differentiation, and may provide useful information for controlling hBM-MSCs growth and differentiation for tissue engineering.
Mesenchymal stem cells (MSCs) can self-renew, are multipotent cells and can differentiate into many different cells. They are important cell souece for tissue engineering and cell therapy. So far, little is known about how MSC growth and differentiation is regulated, and this study tried to gain some insight into this question by identification of genes differentially expressed in human bone marrow mesenchymal stem cells (hBM-MSCs) between early passages and late passages. hBM-MSCs at passage 5 and passage 20 were used as a tester and a driver respetively for subtractive hybridization experiment. PCR product derived from subtractive hybridization was cloned into pGEM-Teasy vector. A total of 117 positive clones were taken for DNA sequencing. DNA sequence of each clone was compared with GenBank database for gene homologes, and 21 different human known genes were identified representing the 117 clones. DNA sequence homologies between each of these 21 genes and their respective known genes were greater than 90%. To confirm the differential expression of these genes, semi-quantitative RT-PCR analysis were performed, and it showed that 6 genes were indeed differentially expressed in hBM-MSCs between early passages and late passages by more than 1 fold. These genes included rab23, ddx21, tnfaip3, nomo2, pabpc3 and cnn3. These genes may play an important role in maitaning and regulating hBM-MSCs growth and differentiation, and may provide useful information for controlling hBM-MSCs growth and differentiation for tissue engineering.
引文
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[26] Sanchez-Ramos J, Song S, Cardozo-pelaze F. Adult bon marrow stromal cells differentiate into neural cells in vitro. Exp Neuro1, 2000, 164(2):247.
[27] Nuttall ME, Patton, Olives DL. Human trabecular bone cells are able to express both osteoblastic and adipocytic phenotype: implications for osteogenic disorders. J Bone Miner Res, 1998, 13(3):371.
[28] Leek, Majumdav Mk, Buyaner D. Human mesenchymal stem cells maintain transgene expression during expansion and differentiation. Mol Ther, 2001, 3(6):857.
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