本文選題：臍帶膠質(zhì) + 人臍帶間充質(zhì)細胞��； 參考：《內(nèi)蒙古農(nóng)業(yè)大學》2011年碩士論文

【摘要】：細胞治療已經(jīng)走過十多年的歷程,起初科學家對免疫細胞進行大量研究,如細胞因子誘導的殺傷細胞(CIK)治療癌癥具有可觀的前景;而目前科學界對人臍帶間充質(zhì)干細胞(Human umbilical cord mesenchymal stem cell, hUC-MSC)的研究較為關(guān)注,由于hUC-MSC比胚胎干細胞(ESC)、骨髓間充質(zhì)干細胞(BM-MSC)等具有更好的臨床應(yīng)用潛能,從而成為人類再生醫(yī)學和組織工程的研究熱點。 本研究目的是探索更有效的hUC-MSC分離培養(yǎng)方法和進一步研究hUC-MSC生物學特性,通過建立優(yōu)化的實驗方法,能為再生醫(yī)學科研和臨床應(yīng)用提供操作規(guī)范標準和參考依據(jù)。方法:實驗隨機選取足月順產(chǎn)健康胎兒臍帶,采取手工機械分離法和3種酶組合消化法從臍帶膠質(zhì)中釋放人臍帶間充質(zhì)細胞;并用優(yōu)化完全培養(yǎng)基純化培養(yǎng);MTT法檢測各代臍帶間充質(zhì)細胞生長增殖活性并繪制生長曲線;流式細胞術(shù)檢測臍其生長周期和表面標志物;RT-PCR擴增基因P53、C-myc和OCT-4;結(jié)果:培養(yǎng)臍帶間充質(zhì)細胞形態(tài)為梭形、呈纖維樣,并出現(xiàn)流水狀或漩渦樣生長;生長增殖速度較快,第7代臍帶間充質(zhì)細胞指數(shù)倍增時間小于30h;80%以上細胞處于G0/G1期;培養(yǎng)臍帶間充質(zhì)細胞均一穩(wěn)定高表達CD29、CD70、CD105、CD90和CD44,而幾乎不表達CD45、CD34和HLA-DR;P53基因在各代臍帶間充質(zhì)細胞均有表達,C-myc基因在第4代和第18代出現(xiàn)明顯表達,OCT-4基因在不同代數(shù)均出現(xiàn)表達;結(jié)論:1)1h可分離30cm臍帶膠質(zhì),獲得的大量臍帶間充質(zhì)細胞(9×10~4/cm),經(jīng)培養(yǎng)檢測證明具有hUC-MSC的形態(tài)特征、增殖活性,表面抗原一致,是原始的hUC-MSC;2)通過RT-PCR檢測各代臍帶間充質(zhì)細胞的P53、C-myc和OCT-4基因,發(fā)現(xiàn)培養(yǎng)10代以內(nèi)hUC-MSC狀態(tài)較正常。本研究意義:提示在臨床應(yīng)用hUC-MSC時,對其癌基因和抑癌基因全面檢測以及衰老凋亡系統(tǒng)檢測很有必要;本實驗可為短期培養(yǎng)擴增出臨床治療所需要安全可靠的MSC提供研究基礎(chǔ),可為避免hUC-MSC體內(nèi)移植治療產(chǎn)生致瘤性提供參考依據(jù)。
[Abstract]:Cell therapy has gone through more than ten years. At first, scientists carried out a lot of research on immune cells, such as cytokine induced killer cells (CIK) treatment of cancer has considerable prospects; Human umbilical cord mesenchymal stem cell, hUC-MSC (human umbilical cord mesenchymal stem cell) has been paid more attention to by the scientific community at present. Because hUC-MSC has better clinical application potential than embryonic stem cell (ESC), bone marrow mesenchymal stem cell (BM-MSC), and so on, human umbilical cord mesenchymal stem cell (hUC-MSC) has better clinical application potential. Therefore, it has become the research hotspot of human regenerative medicine and tissue engineering. The purpose of this study was to explore a more effective method for the isolation and culture of hUC-MSC and to further study the biological characteristics of hUC-MSC. By establishing an optimized experimental method, it can provide the standard and reference for the scientific research and clinical application of regenerative medicine. Methods: human umbilical cord mesenchymal cells were released from cord glia by manual mechanical separation and three enzyme digestion methods. The growth and proliferation activity of umbilical cord mesenchymal cells was determined by MTT method and the growth curve was drawn. Flow cytometry was used to detect the growth cycle and surface marker of umbilical cord by RT-PCR. Results: the morphology of cultured umbilical cord mesenchymal cells was fusiform, fibrous, and the growth of umbilical cord mesenchymal cells was like water or whirlpool. The doubling time of the seventh passage mesenchymal cell index was less than 30 h and more than 80% of the cells were in G _ 0 / G _ 1 phase. In cultured umbilical cord mesenchymal cells, the expression of CD29, CD70, CD105, CD90 and CD44, and almost no expression of CD45, CD34 and HLA-DRN p53 genes in all generations of umbilical cord mesenchymal cells, were significantly expressed in the 4th and 18th generation of umbilical cord mesenchymal cells, and OCT-4 genes were expressed in different generations. Conclusion 30cm cord glia can be isolated from 30cm for 1 hour. A large number of umbilical cord mesenchymal cells (9 脳 10 ~ (4) / cm ~ (-1) 路cm ~ (-1) were obtained. The morphological characteristics, proliferative activity and surface antigen of HUC-MSC were confirmed by culture. The P53C-myc and OCT-4 genes of umbilical cord mesenchymal cells were detected by RT-PCR. It was found that the status of hUC-MSC was normal within 10 generations. The results indicate that it is necessary to detect the oncogenes and tumor suppressor genes and the aging apoptosis system in the clinical application of hUC-MSC, and this study can provide a research basis for the amplification of safe and reliable MSC for clinical treatment in short term culture. It can provide reference for avoiding tumorigenicity of hUC-MSC transplantation in vivo.
【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】：2011
【分類號】：R329

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本文編號：2031522