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为探讨成人骨髓分离培养神经干细胞的可行性和有效性 ,无菌条件下行骨穿 ,梯度密度离心分离获取成人骨髓源细胞 ,以“CYTOKINE·神经干细胞培养液”培养 ,确定神经干细胞的最佳体外生存环境。以细胞克隆方法判断神经干细胞增殖情况 ;以NESTIN、NSE及GFAP免疫细胞化学方法分别鉴定神经干细胞、神经元和神经胶质细胞。细胞培养所涉及的细胞因子主要有GDNF(2 0ng/ml)、LIF(10ng/ml)和RA(0 5 μg/ml)等。结果发现 ,成人骨髓源细胞在相应培养条件下快速分裂增殖为大而圆并含粗大胞质颗粒的神经干细胞 ,后者形成细胞球 (或称“神经球”) ,该细胞球表达特殊的神经干细胞NESTIN抗原 ;若进一步将这些细胞球分离成单细胞并重新以克隆密度培养 ,单个的细胞又很快形成新的神经球。神经干细胞球进一步分化 ,可见到有的细胞胞体增大并出芽 ,逐渐发育成为较成熟的长突起细胞 ,长突起相互连接、交织成网并建立有神经纤维样联系 ,表达NSE或GFAP抗原。说明成年人骨髓在一定条件诱导下可以生成神经干细胞 ;用人骨髓组织诱导神经干细胞作为种子细胞具有可行性和有效性
To explore the feasibility and effectiveness of adult bone marrow-derived neural stem cells isolated from bone marrow, bone marrow-derived and gradient-density centrifugation was performed under sterile conditions to obtain adult bone marrow-derived cells. Cultured with “CYTOKINE · Neural Stem Cell Culture Medium”, the best in vitro neural stem cells living environment. The proliferation of neural stem cells was evaluated by cell clone method. Neural stem cells, neurons and glial cells were identified by NESTIN, NSE and GFAP immunocytochemistry. The main cytokines involved in cell culture are GDNF (20 ng / ml), LIF (10 ng / ml) and RA (0 5 μg / ml). The results showed that adult bone marrow-derived cells rapidly divide and proliferate into large round and thick cytoplasmic granules of neural stem cells under the corresponding culture conditions, which form a cell spheroid (or “neurosphere”), which expresses a special nerve Stem cells NESTIN antigen; If these cells were further separated into single cells and re-clone density in culture, the individual cells quickly form new neurospheres. Further differentiation of neural stem cells, we can see some cells increased and budding cell bodies, and gradually developed into more mature long protuberant cells, long protrusions connected to each other, interwoven into a network and the establishment of a nerve fiber-like connection, the expression of NSE or GFAP antigen. It shows that adult bone marrow can generate neural stem cells under certain conditions; it is feasible and effective to induce neural stem cells as seed cells by human bone marrow