源于成人骨髓神经干细胞诱导分化的实验研究(英文)

Experimental study of the differentiation induction of adult human bone marrow-derived neural stem cells

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。

AIM:To study the feasibilit y of isolatin g and culturing neural stem cells(NSCs )from adult human bone marr ow for pote ntial clinical application of NSCs autotransplantation for treating cerebral degenerative diseases.METHODS:Ilium puncture was performed in 20ad ult voluntary donors of either genders(aged between 28and 48years)to obtai n the bone marrow specimens,from which bone marrow stromal cells were harvested by means o f density gradient centrifugation an d cultured in'Cytokine·NSCs m edium(a patent medium prepared by our lab)to ascertain the optimal in vitro culture conditions of the cells.The proliferation of NSCs was confirmed by obs erving the cell clone formation,and the NSCs,neurons and glial cells were iden tified by immunocytochemi cal detection of the expressions of nestin,neuron-sp ecific endase(NSE),and glial fibrillary acidic protein(GFAP)respectively.T he cytokines employed in the cell culture mainly in-cluded brain-derived neuro trophic factor(BDNF),leukemia inhibitory factor(LIF,10μg /L)and retino ic acid(RA,0.5mg /L).RESULTS:When proper culture condition was sa tisfi ed,the bone marrow stromal cells proliferated rapidly and differentiated into N SCs,some o f which that contained rough and large cytop lasmic granules merged into islet-like spherical cell cluster(nerve bulb)which were positive of nest in.After the cell clusters were separated into single cells for further culture ,the i slet-like pellets again appeared.Further differentiation of the NSCs c lusters ga ve rise to the formation of small buds in some cells that developed into the long projects with the potential to connect with each other.The cells with lon g projects were positive for NSE or GFP expression.