人毛囊间充质干细胞重编程诱导为iPS细胞的研究

发布时间：2018-01-29 22:08

本文关键词： 诱导性多潜能干细胞人毛囊间充质干细胞胚胎干细胞　出处：《吉林大学》2012年博士论文　论文类型：学位论文

【摘要】：干细胞的来源和数量一直是干细胞组织工程研究领域的重点和难点。胚胎干细胞（ESCs）的获得及其向多种组织细胞诱导的成功，曾给许多认为是不治之症的患者带来生存和康复的希望，但很快受到伦理的困扰而举步维艰；成体干细胞的异体应用虽可回避伦理问题，但又受到免疫排斥和疾病传播风险的限制；iPS细胞的技术突破为倍受争议的人胚胎干细胞（hESCs）的应用提出了新的解决方案，并已经取得一些重大成果，，目前是一旦传递多能性因子的载体以及诱导的低效率问题得到解决，那么重编程靶细胞的选择即成为亟待解决的当务之急。为建立一种来源丰富、取材方便，取材对机体无损害且可重复获得的自体干细胞作为重编程靶细胞的来源，本实验从人毛囊间充质干细胞（hHF-MSCs）的分离、培养、扩增及鉴定入手，并以hHF-MSCs为靶细胞，进行了去分化重编程为iPS细胞的实验研究。通过直接拔取人头发的方式，首次在人毛囊组织中发现具有多向分化潜能的干细胞。经细胞表面抗原以及中胚层细胞谱系等多向分化能力的检测，证实了我们获得的毛囊源性干细胞的间充质性质，并确定为人毛囊间充质干细胞（hHF-MSCs），这不仅是本研究的重要发现，也为后续实验奠定了坚实的基础。随后我们研究组以hHF-MSCs作为研究的靶细胞，通过Yamanaka因子（Oct4，Sox2，c-Myc，Klf4）的导入，首次将其成功的重编程为iPS细胞。通过碱性磷酸酶染色，人胚胎干细胞特异性细胞表面抗原SSEA-4，SSEA-3，Tra-1-60，Tra-1-81等检测，内源性多能性基因Oct4endo，Sox2endo，Nanog，Cripto，FoxD3，Lin28等表达是否上调的检测，核型分析以及免疫缺陷小鼠体内畸胎瘤形成实验，证实了hHF-MSC-derived iPSCs具有和hESCs相同的特点，从而首次将hHF-MSCs重编程为iPS细胞获得成功，为hHF-MSC-derived iPSCs的进一步应用提供了重要的实验基础。综上所述，本实验通过hHF-MSCs体外分离培养、扩增的技术体系的建立和将hHF-MSCs重编程为iPS细胞的成功，分别获得了hHF-MSCs和hHF-MSC-derivediPSCs。两种细胞的获得不仅对干细胞来源等的基础研究是一重大突破，也可以用hHF-MSC-derived iPSCs代替hESCs进行组织发生、药物筛选、再生医学等的许多重大基础研究，也为组织工程材料的构建提供新的种子细胞来源。同时，为应用iPSCs进行疾病的个体化治疗提供新的途径。
[Abstract]:The origin and quantity of stem cells have been the focus and difficulty in stem cell tissue engineering. The acquisition of embryonic stem cells (ESCs) and its successful induction to a variety of tissue cells. It has brought hope of survival and rehabilitation to many patients who are thought to be incurable, but it is quickly troubled by ethics. Although allogeneic application of adult stem cells can avoid ethical problems, it is restricted by immune rejection and risk of disease transmission. The technical breakthrough of iPS cells has proposed a new solution for the controversial application of human embryonic stem cells (hESCs), and has achieved some significant results. At present, once the carrier of the transfer of pluripotent factor and the problem of induced inefficiency are solved, the choice of reprogrammed target cells becomes an urgent task to be solved. The autologous stem cells which have no damage to the body and can be obtained repeatedly as the source of reprogrammed target cells were isolated, cultured, amplified and identified from human hair follicle mesenchymal stem cells (hHF-MSCs). The dedifferentiation and reprogramming of hHF-MSCs cells into iPS cells were studied. Through the direct extraction of human hair, stem cells with multidirectional differentiation potential were first found in human hair follicles. The multidirectional differentiation ability of stem cells was detected by cell surface antigen and mesodermal cell lineage. We confirmed the mesenchymal properties of hair follicle-derived stem cells and identified them as human hair follicle mesenchymal stem cells. This is not only an important discovery in this study. It also laid a solid foundation for the follow-up experiment. Then we used hHF-MSCs as the target cell of the study, and used Yamanaka factor Oct4, Sox2nc-Myc. Klf4) was successfully reprogrammed into iPS cells for the first time. By alkaline phosphatase staining, human embryonic stem cell specific cell surface antigen SSEA-4 / SSEA-3 was obtained. The detection of Tra-1-60, Tra-1-81, et al., showed that the endogenous pluripotent gene Oct4endo1 Sox2endo1 Nanoga Criptosiae FoxD3 was detected. Detection of upregulation of Lin28 expression, karyotype analysis and teratoma formation in immunodeficient mice. It is proved that hHF-MSC-derived iPSCs has the same characteristics as hESCs, so hHF-MSCs reprogramming as iPS cell is successful for the first time. It provides an important experimental basis for the further application of hHF-MSC-derived iPSCs. To sum up, this experiment was carried out through the isolation and culture of hHF-MSCs in vitro, the establishment of amplification technique system and the success of reprogramming hHF-MSCs into iPS cells. The acquisition of hHF-MSCs and hHF-MSC-derived PSCs. is not only a breakthrough in the basic research of stem cell sources. HHF-MSC-derived iPSCs can also be used instead of hESCs to carry out many important basic research on histogenesis, drug screening, regenerative medicine and so on. It also provides a new source of seed cells for the construction of tissue engineering materials and a new way for individualized treatment of disease with iPSCs.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329.28

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