人皮肤成纤维细胞诱导多潜能干细胞的研究

发布时间：2018-05-15 05:36

本文选题：多潜能干细胞(iPSCs) + 基因治疗　；参考：《中南大学》2011年硕士论文

【摘要】：目的：通过异位表达某些转录因子,可以将体细胞重编程成诱导性多潜能干细胞(iPSCs),它能避开ES细胞带来的伦理问题,减少免疫排斥,给自体化干细胞基因治疗的临床应用提供了可能性,为遗传性疾病体外研究提供了一个很好的平台。方法：目前有多种方法可诱导获得iPS细胞,包括利用各种病毒载体、纯化的重组蛋白质、修饰的RNA分子等。同时研究发现多种小分子化合物也可促进iPS的产生,如Vc能加速基因表达水平的改变,促进前体iPS细胞转变成完全重编程的iPS细胞；VPA在重编程过程中,可上调ES特异性基因的表达,下调MEF特异性基因的表达。在本研究中,我们利用逆转录病毒介导的转染系统将Oct4,Sox2,c-Myc和Klf4这4个转录因子导入人皮肤成纤维细胞(HDF),同时在诱导过程中添加Vc和VPA,最终获得iPS细胞。然后,通过TRA-1-60/TRA-1-81活细胞染色,机械法挑取阳性克隆传代培养。采取形态学、碱性磷酸酶染色以及干细胞表面多潜能标志物染色的方法对传代后的克隆进行初步鉴定。结果：(1)用脂质体Lipofectamine2000将逆转录病毒载体及包装质粒共同转染293T细胞,可获得较高质量的逆转录病毒；(2)诱导出的克隆在形态上与hES细胞相似,外源基因GFP不表达,经过活细胞染色发现其TRA-1-60/TRA-1-81有表达,挑取上述克隆传代后,AP染色结果呈阳性,ES细胞表面标志免疫荧光检测呈阳性。传至P7代,仍然具有ES样克隆形态。结论：利用逆转录病毒系统将人皮肤成纤维细胞重编程为iPS细胞,经初步鉴定可表达多潜能性表面标志物。
[Abstract]:Objective: by ectopic expression of some transcription factors, somatic cells can be reprogrammed into inducible multipotential stem cells, which can avoid the ethical problems caused by es cells and reduce immune rejection. It provides the possibility for the clinical application of autologous stem cell gene therapy and provides a good platform for the in vitro study of genetic diseases. Methods: there are many methods to induce iPS cells, including the use of various virus vectors, purified recombinant proteins, modified RNA molecules and so on. At the same time, it was found that many kinds of small molecular compounds can also promote the production of iPS, such as VC can accelerate the change of gene expression level and promote the transformation of precursor iPS cells into fully reprogrammed iPS cells during the reprogramming process. It can up-regulate the expression of es specific gene and down-regulate the expression of MEF specific gene. In this study, we used a retrovirus-mediated transfection system to transfer the four transcription factors Oct4nSox2Oc-Myc and Klf4 into human skin fibroblasts, and then added VC and VPA during the induction process to obtain iPS cells. Then, the positive clones were subcultured by TRA-1-60/TRA-1-81 staining, and the positive clones were subcultured by mechanical method. The clones were identified by morphology, alkaline phosphatase staining and multipotential marker staining on stem cell surface. Results using liposome Lipofectamine2000 to co-transfect retrovirus vector and packaging plasmid into 293T cells, a high quality retroviral vector was obtained. The clone induced by the recombinant plasmid was similar to that of hES cells in morphology, and the foreign gene GFP was not expressed. The expression of TRA-1-60/TRA-1-81 was found by living cell staining, and the AP staining was positive after the above clones were selected. The surface markers of es cells were detected positive by immunofluorescence. After transmission to P7 generation, it still had es like clone morphology. Conclusion: human skin fibroblasts were reprogrammed into iPS cells by retrovirus system, and multipotential surface markers could be expressed.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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