小鼠髓母细胞瘤干细胞分离培养及干细胞标记物的分析

目的 旨在建立自发髓母细胞瘤模型小鼠肿瘤干细胞分离培养方法,观察其在体外形成克隆的能力,对可能的肿瘤干细胞标志分子CD44、CD133和CD15进行流式细胞术分析鉴定.方法 将小鼠髓母细胞瘤组织通过温和消化液消化分离成单细胞悬液,于干细胞培养基中培养.计算其细胞球形成率.于含血清培养基中培养观察分化能力.利用流式细胞术对其表面可能的干细胞表面标记分子进行分析鉴定.结果 从模型小鼠髓母细胞瘤组织中成功分离培养髓母细胞瘤原代细胞,原代细胞可形成具有高度的自我更新和增殖能力的细胞球;细胞球可在含血清培养基中贴壁分化成为神经元样细胞;干细胞表面标记分子流式细胞术分析表明髓母细胞瘤干细胞中CD44表达较高,CD133及CD15的表达无差异或者降低.结论 髓母细胞瘤肿瘤细胞中存在一定量的具有自我更新增殖能力、高表达CD44的肿瘤干细胞,并能在体外将其分离培养、连续传代及诱导发生分化.

Mice Medulloblastoma Stem Cells Isolating and Culturing and Its Biomarker Analysis

Objective This study aimed to optimize the method of isolating and culturing tumor stem cells in spontaneous isolating and culturing tumor stem cells in spontaneous medulloblastoma(MB)model mice model mice,and observe the ability of these stem cells to form clone in vitro,and analyze the potential tumor stem cell markers including CD44,CD133 and CD15 through flow cytometry.Methods Tissue of medulloblastoma were dissociated into single cells,which were then cultured in stem cell culture medium.Then we calculated the rate of stem cell sphere formation rate and observed differentiative capacity of stem cells when cultured in serum medium.Finally,the flow cytometry analysis was employed to identify the potential surface marker molecules of stem cells.Results Primary medulloblastoma cells was successfully isolated from the medulloblastoma model mice,which then transform into cell spheroids with advanced self-renewing and proliferating abilities.The cells spheroids could went through the adherence differentiation in serum medium and transform into neuron-like cells.The flow cytometry analysis of stem cells surface markers showed a high expression of CD44 in medulloblastoma,whereas the expressions of CD133 and CD15 are relatively low or showed no variations.Conclusion This study confirms the existence of certain amount of high expression CD44 tumor stem cells in medulloblastoma,which possess the ability to self-renew and proliferate.These tumor stem cells could be isolated and cultured in vitro,grow in iterations,and be induced to differentiate.