胚鼠室管膜神经干细胞体外诱导分化实验研究

目的探寻胚鼠室管膜分离培养及诱导分化为神经干细胞(NSCs)的可行性及规律性,为进一步寻找非神经组织性NSCs种子源提供阳性参照。方法将分离的胚脑室管膜组织以神经干细胞培养液孵育,确定神经干细胞的最佳体外生存环境,细胞培养所涉及的细胞因子主要有:EGF、bFGF和LIF(分别为20 ng/ml)。以细胞克隆方法判断神经干细胞增殖;以镜下细胞形态初步判断神经干细胞分化。结果胚鼠室管膜源性神经干细胞在相应培养条件下呈现出神经干细胞快速增殖,形成由多细胞组成的细胞球(神经球);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。神经干细胞连续培养可进一步有小芽形成并发育成突起状结构、建立神经纤维联系,其中有的胞体增大,逐渐发育为较成熟的长突起细胞,长突起相互连接、交织成网。结论由胚鼠室管膜分离培养神经干细胞是可行的;神经干细胞在发育分化过程,基本上遵循着游离神经干细胞、神经细胞球、分化神经元/胶质细胞的生长规律,显现出神经干细胞有别于一般神经细胞的增殖及多分化特性。

Experimental study on in vitro inducement differentiation of neural stem cells from fetal rat ependyma

Objective To explore the feasibility and regularity of neural stem cells (NSCs) culture and inducement differentiation from fetal rat ependyma, and offer the positive control for searching the NSCs seed cells outside the nerve tissue. Methods The cells of fetal rat ependyma were cultured in NSCs medium in order to ascertain the optimal survival conditions of NSCs in vitro. The involved cytokines mainly included EGF, bFGF and LIF (20 ng/ml respectively). The proliferation of NSCs was confirmed by formation of cell clones. The differentiation of NSCs was primarily estimated by observing the cellsmorphology under a microscope. Results The NSCs derived from the fetal rat ependyma were observed to rapidly proliferate to form some cellular spheres or neural spheres consisting of NSCs. Having been separated into single cells and then plated, the islets-shaped cellular spheres were found to appear again. Continuous incubation of the NSCs in NSCs-medium could lead to their further proliferation. Some of them became large enough to grow into cell-body, and some small buds were formed from several NSCs bodies and then developed into the long projects that might be connected with each other. Conclusion It is feasible for the fetal rat ependyma to be induced into NSCs. The gradual developmental process of NSCs follows the rule of growing from single NSCs, to neural sphere and at last to the differentiated neurons, through which the NSCs manifest themselves with characteristics of proliferation and differentiation different from the general neurons.