本文關(guān)鍵詞： 誘導性多潛能干細胞 人毛囊間充質(zhì)干細胞 胚胎干細胞　出處：《吉林大學》2012年博士論文　論文類型：學位論文

【摘要】：干細胞的來源和數(shù)量一直是干細胞組織工程研究領(lǐng)域的重點和難點。胚胎干細胞（ESCs）的獲得及其向多種組織細胞誘導的成功，曾給許多認為是不治之癥的患者帶來生存和康復(fù)的希望，但很快受到倫理的困擾而舉步維艱；成體干細胞的異體應(yīng)用雖可回避倫理問題，但又受到免疫排斥和疾病傳播風險的限制；iPS細胞的技術(shù)突破為倍受爭議的人胚胎干細胞（hESCs）的應(yīng)用提出了新的解決方案，并已經(jīng)取得一些重大成果，，目前是一旦傳遞多能性因子的載體以及誘導的低效率問題得到解決，那么重編程靶細胞的選擇即成為亟待解決的當務(wù)之急。為建立一種來源豐富、取材方便，取材對機體無損害且可重復(fù)獲得的自體干細胞作為重編程靶細胞的來源，本實驗從人毛囊間充質(zhì)干細胞（hHF-MSCs）的分離、培養(yǎng)、擴增及鑒定入手，并以hHF-MSCs為靶細胞，進行了去分化重編程為iPS細胞的實驗研究。 通過直接拔取人頭發(fā)的方式，首次在人毛囊組織中發(fā)現(xiàn)具有多向分化潛能的干細胞。經(jīng)細胞表面抗原以及中胚層細胞譜系等多向分化能力的檢測，證實了我們獲得的毛囊源性干細胞的間充質(zhì)性質(zhì)，并確定為人毛囊間充質(zhì)干細胞（hHF-MSCs），這不僅是本研究的重要發(fā)現(xiàn)，也為后續(xù)實驗奠定了堅實的基礎(chǔ)。隨后我們研究組以hHF-MSCs作為研究的靶細胞，通過Yamanaka因子（Oct4，Sox2，c-Myc，Klf4）的導入，首次將其成功的重編程為iPS細胞。通過堿性磷酸酶染色，人胚胎干細胞特異性細胞表面抗原SSEA-4，SSEA-3，Tra-1-60，Tra-1-81等檢測，內(nèi)源性多能性基因Oct4endo，Sox2endo，Nanog，Cripto，F(xiàn)oxD3，Lin28等表達是否上調(diào)的檢測，核型分析以及免疫缺陷小鼠體內(nèi)畸胎瘤形成實驗，證實了hHF-MSC-derived iPSCs具有和hESCs相同的特點，從而首次將hHF-MSCs重編程為iPS細胞獲得成功，為hHF-MSC-derived iPSCs的進一步應(yīng)用提供了重要的實驗基礎(chǔ)。 綜上所述，本實驗通過hHF-MSCs體外分離培養(yǎng)、擴增的技術(shù)體系的建立和將hHF-MSCs重編程為iPS細胞的成功，分別獲得了hHF-MSCs和hHF-MSC-derivediPSCs。兩種細胞的獲得不僅對干細胞來源等的基礎(chǔ)研究是一重大突破，也可以用hHF-MSC-derived iPSCs代替hESCs進行組織發(fā)生、藥物篩選、再生醫(yī)學等的許多重大基礎(chǔ)研究，也為組織工程材料的構(gòu)建提供新的種子細胞來源。同時，為應(yīng)用iPSCs進行疾病的個體化治療提供新的途徑。
[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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R329.28

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