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  本文選題：羊膜 + 間充質(zhì)干細(xì)胞　； 參考：《昆明醫(yī)學(xué)院》2010年碩士論文

【摘要】： 目的:探討人羊膜來源的間充質(zhì)干細(xì)胞(mesenchymal stem cells,MSCs)在體外對(duì)造血干細(xì)胞(hematopoietic stem cells,HSCs)的擴(kuò)增是否有支持作用,以促進(jìn)羊膜間充質(zhì)干細(xì)胞(amniotic mesenchymal stem cells,AMSCs)的臨床應(yīng)用,尤其為臨床移植前擴(kuò)增HSCs數(shù)量,以及MSCs和HSCs共移植、提高干細(xì)胞移植成功率提供實(shí)驗(yàn)依據(jù)。 方法:利用成熟的MSCs培養(yǎng)條件,從足月分娩的人羊膜中分離培養(yǎng)AMSCs;用密度梯度離心法分離臍血單個(gè)核細(xì)胞:用免疫磁珠分選技術(shù)分選其中CD34~+細(xì)胞:分別用臍血單個(gè)核細(xì)胞和CD34~+細(xì)胞與AMSCs共培養(yǎng),連續(xù)培養(yǎng)四周,每周計(jì)懸浮細(xì)胞濃度,并把它們分別與骨髓MSCs共培養(yǎng)作為對(duì)照。共培養(yǎng)兩周后,利用HSCs定向分化的潛能,采用集落形成實(shí)驗(yàn),把擴(kuò)增的臍血單個(gè)核細(xì)胞和CD34~+細(xì)胞分別接種至甲基纖維素半固體培養(yǎng)基中,14-16天后根據(jù)典型形態(tài)特征計(jì)CFU-GM、BFU-E、CFU-E和CFU-GEMM集落數(shù)。結(jié)果均以(?)±S表示,各組均數(shù)間差異性檢驗(yàn)用單因素方差分析。 結(jié)果:從人羊膜分離培養(yǎng)出的干細(xì)胞在形態(tài)和表型上均符合MSCs的特征,細(xì)胞呈梭形和多角形。流式分析顯示,AMSCs高表達(dá)CD29、CD44、CD105,不表達(dá)CD31、CD34、CD45、pan-CK等內(nèi)皮、造血及上皮細(xì)胞表型,HLA-DR呈陰性。AMSCs可促進(jìn)人臍血單個(gè)核細(xì)胞和CD34~+細(xì)胞擴(kuò)增,擴(kuò)增后的兩者在甲基纖維素半固體培養(yǎng)基中能夠形成造血祖細(xì)胞集落,其造血支持作用與骨髓MSCs相似,兩者對(duì)比無明顯差異。而無MSCs支持的臍血單個(gè)核細(xì)胞和CD34~+細(xì)胞,其擴(kuò)增能力和集落形成能力明顯減弱。 結(jié)論:用組織塊培養(yǎng)法可以從人羊膜中成功分離培養(yǎng)出MSCs;AMSCs體外具有與骨髓MSCs相似的造血支持作用,有可能為HSCs體外擴(kuò)增及臨床MSCs與HSCs聯(lián)合移植、提高HSCs移植成功率提供一種新的、更加理想的MSCs新來源。
[Abstract]:Objective: to investigate whether mesenchymal stem cells (MSCs) derived from human amniotic membrane can support the expansion of hematopoietic stem cells (HSCs) in vitro, so as to promote the clinical application of amniotic mesenchymal stem cells / amniotic cells. Especially, it provides experimental basis for increasing the number of HSCs before transplantation and co-transplantation of MSCs and HSCs to improve the success rate of stem cell transplantation. Methods: using mature MSCs culture conditions, AMSCs were isolated from human amniotic membrane during term delivery, cord blood mononuclear cells were isolated by density gradient centrifugation, CD34 ~ cells were separated by immunomagnetic bead sorting technique: cord blood mononuclear cells and CD34 ~ cells were co-cultured with AMSCs for four weeks, respectively. Suspension cell concentration was measured weekly and co-cultured with bone marrow MSCs as control. After two weeks of co-culture, colony forming experiments were carried out using the potential of HSCs directional differentiation. The expanded cord blood mononuclear cells and CD34~ cells were inoculated into methylcellulose semisolid medium for 14-16 days. The colony numbers of CFU-GMU BFU-EU CFU-E and CFU-GEMM were calculated according to the typical morphological characteristics. The results were all expressed in the form of + S, and the analysis of variance was used to test the difference of the mean of each group by single factor analysis of variance (ANOVA). Results: the stem cells isolated from human amniotic membrane were morphologically and phenotypically consistent with the characteristics of MSCs, and the cells were fusiform and polygonal. Flow cytometry showed that AMSCs overexpressed CD29, CD44, CD105, and did not express CD31, CD34, CD45, pan-CK and other endothelium. HLA-DR negative phenotype of hematopoietic and epithelial cells could promote the expansion of mononuclear cells and CD34~ cells in human umbilical cord blood. The hematopoietic progenitor cell colony was formed in methyl cellulose semisolid medium, and the hematopoietic supporting effect was similar to that of bone marrow MSCs, but there was no significant difference between them. However, the ability of expansion and colony formation of cord blood mononuclear cells and CD34 ~ cells without MSCs support was significantly decreased. Conclusion: MSCs can be successfully isolated from human amniotic membrane by tissue mass culture method and have similar hematopoietic support effect to bone marrow MSCs in vitro, which may be HSCs amplification in vitro and clinical MSCs combined with HSCs transplantation. Increasing the success rate of HSCs transplantation provides a new and more ideal source of MSCs.
【學(xué)位授予單位】：昆明醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

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本文編號(hào)：1953063