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

【摘要】：細(xì)胞治療已經(jīng)走過(guò)十多年的歷程,起初科學(xué)家對(duì)免疫細(xì)胞進(jìn)行大量研究,如細(xì)胞因子誘導(dǎo)的殺傷細(xì)胞(CIK)治療癌癥具有可觀的前景;而目前科學(xué)界對(duì)人臍帶間充質(zhì)干細(xì)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(xì)胞(ESC)、骨髓間充質(zhì)干細(xì)胞(BM-MSC)等具有更好的臨床應(yīng)用潛能,從而成為人類(lèi)再生醫(yī)學(xué)和組織工程的研究熱點(diǎn)。 本研究目的是探索更有效的hUC-MSC分離培養(yǎng)方法和進(jìn)一步研究hUC-MSC生物學(xué)特性,通過(guò)建立優(yōu)化的實(shí)驗(yàn)方法,能為再生醫(yī)學(xué)科研和臨床應(yīng)用提供操作規(guī)范標(biāo)準(zhǔn)和參考依據(jù)。方法:實(shí)驗(yàn)隨機(jī)選取足月順產(chǎn)健康胎兒臍帶,采取手工機(jī)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(xì)胞;并用優(yōu)化完全培養(yǎng)基純化培養(yǎng);MTT法檢測(cè)各代臍帶間充質(zhì)細(xì)胞生長(zhǎng)增殖活性并繪制生長(zhǎng)曲線(xiàn);流式細(xì)胞術(shù)檢測(cè)臍其生長(zhǎng)周期和表面標(biāo)志物;RT-PCR擴(kuò)增基因P53、C-myc和OCT-4;結(jié)果:培養(yǎng)臍帶間充質(zhì)細(xì)胞形態(tài)為梭形、呈纖維樣,并出現(xiàn)流水狀或漩渦樣生長(zhǎng);生長(zhǎng)增殖速度較快,第7代臍帶間充質(zhì)細(xì)胞指數(shù)倍增時(shí)間小于30h;80%以上細(xì)胞處于G0/G1期;培養(yǎng)臍帶間充質(zhì)細(xì)胞均一穩(wěn)定高表達(dá)CD29、CD70、CD105、CD90和CD44,而幾乎不表達(dá)CD45、CD34和HLA-DR;P53基因在各代臍帶間充質(zhì)細(xì)胞均有表達(dá),C-myc基因在第4代和第18代出現(xiàn)明顯表達(dá),OCT-4基因在不同代數(shù)均出現(xiàn)表達(dá);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(xì)胞(9×10~4/cm),經(jīng)培養(yǎng)檢測(cè)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(guò)RT-PCR檢測(cè)各代臍帶間充質(zhì)細(xì)胞的P53、C-myc和OCT-4基因,發(fā)現(xiàn)培養(yǎng)10代以?xún)?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(shí),對(duì)其癌基因和抑癌基因全面檢測(cè)以及衰老凋亡系統(tǒng)檢測(cè)很有必要;本實(shí)驗(yàn)可為短期培養(yǎng)擴(kuò)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(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 呂璐璐;劉擁軍;許貞書(shū);王彤;張浪輝;朱雄鵬;梁琳慧;王晗;陳志哲;韓忠朝;;臍帶源間充質(zhì)干細(xì)胞的分離和生物學(xué)性狀[J];福建醫(yī)科大學(xué)學(xué)報(bào);2006年02期

2 陶然;韓焱福;柴家科;;人臍帶組織間充質(zhì)干細(xì)胞研究進(jìn)展及應(yīng)用前景[J];軍事醫(yī)學(xué)科學(xué)院院刊;2010年03期

3 蔡子微;干細(xì)胞與生物自保護(hù)[J];牡丹江醫(yī)學(xué)院學(xué)報(bào);2002年06期

4 陳新;華建媛;石慶之;;人臍帶間充質(zhì)干細(xì)胞對(duì)再生障礙性貧血T細(xì)胞的調(diào)節(jié)作用[J];中國(guó)組織工程研究與臨床康復(fù);2009年40期

5 唐佩弦;;干細(xì)胞基礎(chǔ)研究進(jìn)展[J];醫(yī)學(xué)研究雜志;2007年06期

6 ;Human umbilical cord Wharton's Jelly-derived mesenchymal stem cells differentiation into nerve-like cells[J];Chinese Medical Journal;2005年23期

，

本文編號(hào)：2031522