【摘要】： 目的:國內(nèi)外多項(xiàng)研究初步表明,人骨髓間充質(zhì)干細(xì)胞(hMSCs)具有多向分化潛能,已有實(shí)驗(yàn)證明在體內(nèi)、體外不同細(xì)胞因子作用下,可以誘導(dǎo)分化為骨、軟骨和脂肪細(xì)胞,參與組織更新、損傷的修復(fù)和器官的重建。本課題將通過體外培養(yǎng)hMSCs ,并通過體外誘導(dǎo)使其向血管內(nèi)皮細(xì)胞方向分化,使目的細(xì)胞具有內(nèi)皮細(xì)胞樣特性,旨在為臨床上骨髓間充質(zhì)干細(xì)胞移植治療缺血性心腦血管疾病以及為血管組織工程提供新的種子細(xì)胞來源。 方法:通過密度梯度離心法分離出成人骨髓單個(gè)核細(xì)胞,接著經(jīng)貼壁法獲取hMSCs ,體外擴(kuò)增培養(yǎng)并通過流式細(xì)胞儀對(duì)其進(jìn)行鑒定。在hMSCs生長培養(yǎng)基(DMEM/F12)中加入血管內(nèi)皮細(xì)胞生長因子(VEGF)、堿性成纖維細(xì)胞生長因子(bFGF),定向誘導(dǎo)hMSCs向血管內(nèi)皮細(xì)胞分化,觀察細(xì)胞形態(tài)學(xué)變化,擴(kuò)增培養(yǎng)后經(jīng)流式細(xì)胞儀進(jìn)行細(xì)胞表型鑒定,熒光顯微鏡下觀察誘導(dǎo)后hMSCs的DiI-ac-LDL攝取能力。 結(jié)果:人骨髓間充質(zhì)干細(xì)胞經(jīng)過24小時(shí)的培養(yǎng),貼壁粘附在培養(yǎng)瓶壁上,換液同時(shí)棄去未貼壁細(xì)胞,繼續(xù)培養(yǎng),細(xì)胞呈克隆樣生長,12天后細(xì)胞生長接近融合。消化傳代,傳代后細(xì)胞生長速度加快,約5-7天后細(xì)胞生長即融合。傳至第四代后,細(xì)胞生長趨于穩(wěn)定,此時(shí)開始進(jìn)行誘導(dǎo)分化。誘導(dǎo)分化24天后,所得細(xì)胞具有攝取Dil-ac-LDL功能。誘導(dǎo)分化同時(shí),分別于第0天、14天和24天用流式細(xì)胞儀檢測細(xì)胞表面的表型CD34、CD45、CD106、HLA-DR ,其表達(dá)結(jié)果如下表: 結(jié)論:本實(shí)驗(yàn)探索了體外誘導(dǎo)人骨髓間充質(zhì)干細(xì)胞分化為血管內(nèi)皮細(xì)胞的可行性。在誘導(dǎo)后24天,經(jīng)流式細(xì)胞儀檢測細(xì)胞的表型,包括CD34, CD45, CD106, HLA-DR,其表達(dá)量明顯增加。與此同時(shí)部分細(xì)胞可攝取Dil-ac-LDL ,提示實(shí)驗(yàn)所得細(xì)胞具有內(nèi)皮細(xì)胞特征。這種誘導(dǎo)分化成的具有內(nèi)皮細(xì)胞表型特征的細(xì)胞在形態(tài)學(xué)上變化不大,仍呈梭形或多角形,提示通過實(shí)驗(yàn)培養(yǎng)并誘導(dǎo)分化后所得到的內(nèi)皮樣細(xì)胞與真正的內(nèi)皮細(xì)胞存在一定差異。
[Abstract]:Objective: many studies at home and abroad have shown that human bone marrow mesenchymal stem cell (hMSCs) has the potential to differentiate into bone, cartilage and adipocytes in vivo and in vitro, and has been proved to be able to differentiate into bone, cartilage and adipocytes by different cytokines in vivo and in vitro. Participate in tissue renewal, repair of injury and reconstruction of organs. In this study, hMSCs was cultured in vitro and induced to differentiate into vascular endothelial cells (VEC) in order to make the target cells have the characteristics of endothelial cells. The aim is to provide a new source of seed cells for clinical transplantation of bone marrow mesenchymal stem cells for the treatment of ischemic cardio-cerebrovascular diseases and vascular tissue engineering. Methods: adult bone marrow mononuclear cells were isolated by density gradient centrifugation and hMSCs was obtained by adherent method. Vascular endothelial growth factor (VEGF),) basic fibroblast growth factor (bFGF),) was added to hMSCs growth medium (DMEM/F12) to induce hMSCs to differentiate into vascular endothelial cells (VEC). The phenotype of hMSCs was identified by flow cytometry, and the ability of DiI-ac-LDL uptake of induced hMSCs was observed under fluorescence microscope. Results: after 24 hours of culture, human bone marrow mesenchymal stem cells adhered to the wall of culture bottle, then abandoned the unadherent cells and cultured on the same time. After 12 days, the cells grew close to fusion. After digestion and passage, the cell growth rate was accelerated, and the cell growth was fused after about 5-7 days. After the fourth passage, the cell growth tended to stabilize, and then began to induce differentiation. After induced differentiation for 24 days, the cells had the function of Dil-ac-LDL uptake. At the same time, the phenotypic CD34,CD45,CD106,HLA-DR on cell surface was detected by flow cytometry on day 0, day 14 and day 24, respectively. Conclusion: this study explored the feasibility of inducing human bone marrow mesenchymal stem cells to differentiate into vascular endothelial cells in vitro. At 24 days after induction, the phenotypes, including the expression of CD34, CD45, CD106, HLA-DR, were detected by flow cytometry. At the same time, some cells could absorb Dil-ac-LDL, which suggested that the cells had the characteristics of endothelial cells. The cells with phenotypic characteristics of endothelial cells, which were induced to differentiate into cells, had little morphological changes, but still appeared fusiform or polygonal. These results suggest that there is a certain difference between the endothelial like cells and the real endothelial cells after cultured and induced differentiation.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

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本文編號(hào)：2395577