本文選題：人臍帶間充質(zhì)干細胞　切入點：細胞分化　出處：《內(nèi)蒙古農(nóng)業(yè)大學》2017年碩士論文　論文類型：學位論文

【摘要】：角質(zhì)形成細胞是皮膚表皮組織中最主要的細胞類型,也是對皮膚損傷進行細胞治療時最重要的細胞類型。間充質(zhì)干細胞可經(jīng)誘導分化為多種細胞,具有極低的免疫原性,在細胞治療領域是一種理想的種子細胞。本研究通過改良的"組織塊二次貼壁法",從臍帶華通氏膠中高效、大量地獲得了人臍帶間充質(zhì)干細胞(human umbilical cord mesenchymal stem cells,hUCMSCs),在特殊的誘導培養(yǎng)基中培養(yǎng)時可分化為多種細胞類型。主要研究結果如下:1.建立了 hUCMSCs細胞系,細胞形態(tài)為長梭形、呈漩渦狀生長。第三代(P3)細胞表達干細胞轉錄因子Sox2、間充質(zhì)干細胞表面標志物基因CD29、CD90和CD105,不表達造血系細胞表面標志物基因CD34、CD45及人白細胞抗原基因HLA-DR。經(jīng)測定,其生長曲線呈"S"型,細胞倍增時間為24.7 h,生長良好、活性很高,染色體為46,XY正常男性核型,遺傳穩(wěn)定。2.建立了 hUCMSCs多向誘導分化培養(yǎng)體系,誘導其向外胚層(神經(jīng)細胞、角質(zhì)形成細胞)和中胚層(成骨細胞)來源的細胞分化,染色鑒定結果均為陽性。3.建立了角質(zhì)形成細胞誘導分化培養(yǎng)體系,hUCMSCs經(jīng)誘導培養(yǎng)后分化為角質(zhì)形成細胞,蛋白質(zhì)水平染色結果顯示其表達細胞角蛋白,基因水平檢測到其特異基因CK19的表達量逐日增高。綜上所述,本實驗創(chuàng)建了 hUCMSCs原代培養(yǎng)的新方法,并成功建立了 hUCMSCs細胞系,獲得的hUCMSCs具有多向分化的能力,同時建立了向角質(zhì)形成細胞誘導分化的培養(yǎng)體系,為間充質(zhì)干細胞的臨床應用尤其是大面積皮膚損傷的細胞治療提供了實驗依據(jù)。
[Abstract]:Keratinocytes are the most important cell types in skin epidermis and the most important cell types in the treatment of skin injury. Mesenchymal stem cells can be induced to differentiate into a variety of cells with very low immunogenicity. It is an ideal seed cell in the field of cell therapy. A large number of human umbilical cord mesenchymal stem cells were obtained from human umbilical cord mesenchymal stem cells, which could differentiate into many cell types in a special induction medium. The main results are as follows: 1. The cell line of hUCMSCs was established, and the cell morphology was fusiform. The third generation of P3 cells expressed stem cell transcription factor Sox2, mesenchymal stem cell surface marker gene CD29, CD90 and CD105, but not hematopoietic cell surface marker gene CD34, CD45 and human leukocyte antigen gene HLA-DR. Its growth curve is "S" type, the cell doubling time is 24.7 h, the cell growth is good, the activity is very high, the chromosome is 46 XY normal male karyotype, the heredity is stable .2.It has established the culture system of hUCMSCs multidirectional induction differentiation, induced it to the ectoderm (nerve cell), The differentiation of keratinocytes from keratinocytes) and mesoderm (osteoblasts) were positive. 3. The culture system of keratinocyte induced differentiation was established and then differentiated into keratinocytes. The results of protein level staining showed that it expressed cytokeratin, and the expression of its specific gene CK19 was increased day by day. In conclusion, a new method of primary culture of hUCMSCs was established, and the hUCMSCs cell line was successfully established. The obtained hUCMSCs has the ability of multi-differentiation, and the culture system of inducing differentiation into keratinocytes has been established, which provides the experimental basis for the clinical application of mesenchymal stem cells, especially for the treatment of extensive skin injury.
【學位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R329.2

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