论文部分内容阅读
目的研究成人骨髓分离培养神经干细胞的可行性,为进一步自体移植治疗脑退行病变的临床应用奠定基础。方法以20例成年男/女性(28～48岁)骨髓自愿捐献者为取材对象,骨穿术后经梯度密度离心分离获取的成人骨髓基质细胞用“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;以巢蛋白(Nestin),神经元特异性烯醇化酶(NSE)和胶质原性纤维酸性蛋白(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。所涉及的细胞因子主要有脑源性神经营养因子(BDNF)、白血病抑止因子(LIF)(10μg/L)和维A酸(RA)(0.5mg/L)等。结果成人骨髓基质细胞在相应培养条件下快速分裂增殖为神经干细胞,并由含粗大胞质颗粒的多个神经干细胞组成岛屿状细胞球(神经球),该细胞球表达特殊的神经干细胞Nestin抗原;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,单个的细胞又很快变成岛屿状神经球。神经干细胞球进一步分化,则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,分化的长突起细胞表达NSE或GFAP。结论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。
Objective To study the feasibility of neural stem cells isolated from adult bone marrow for further clinical application of autologous transplantation for the treatment of brain degeneration. Methods Twenty adult male / female (28- to 48-year-old) bone marrow donor volunteers were enrolled in this study. Adult bone marrow stromal cells obtained by gradient density centrifugation after osteotomy were cultured with “Cytokine · Neural Stem Cell Broth” Stem cells the best in vitro survival environment. The proliferation of neural stem cells was determined by cell cloning method. Neural stem cells, neurons and nerves were identified by immunocytochemistry with nestin, neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP) Glial cells. The main cytokines involved are brain derived neurotrophic factor (BDNF), leukemia inhibitory factor (LIF) (10μg / L) and retinoic acid (RA) (0.5mg / L) and so on. Results Adult bone marrow stromal cells rapidly differentiated into neural stem cells under the corresponding culture conditions. Island spheres (neurospheres) were composed of many neural stem cells containing coarse cytoplasmic granules, which expressed special neural stem cells Nestin antigen. Further separating these cell spheres into single cells and re-culturing them at clonal density, individual cells rapidly become island-shaped neurospheres again. When NSCs were further differentiated, some of the cells could be observed to grow and sprout, gradually developing into more mature long protuberant cells. The long protuberances were connected to each other and were interlaced into meshes. The differentiated long protuberance cells expressed NSE or GFAP. Conclusion Adult bone marrow can generate neural stem cells under certain conditions; it is feasible to use human bone marrow tissue to induce neural stem cells as seed cells.