SD大鼠神经干细胞培养鉴定实验研究

EXPERIMENT ON CULTURE, IDENTIFICATION OF NEURAL STEM CELL FROM SD RATS

为探讨从SD胎鼠室管膜、皮质以及SD成年大鼠脑室下区、皮质分离并鉴定神经干细胞的可行性 ,以从上述部位分离的细胞作为种子细胞来源 ,在本实验室特制“CYTOKINE·神经干细胞培养基”中进行了神经干细胞培养诱导 ,单细胞连续传代培养 ,并分别以Nestin、NSE及GFAP免疫抗体鉴定神经干细胞、神经元和神经胶质细胞。结果发现 ,4种来源的组织细胞在相应培养条件下均有神经干细胞快速增殖 ,并有由多细胞组成的神经球形成 ,经鉴定 ,该细胞球表达特殊的胚胎神经干细胞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进行培养 ,单个的细胞又很快形成神经球。对神经干细胞进行连续培养或加血清传代培养可使其进一步分裂增殖 ,有小芽形成并发育成突起、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 ,经鉴定为神经元及神经胶质细胞。 4种组织来源的细胞中所含有的干细胞数量不同 ,胎鼠较成年鼠更富含神经干细胞 ,室管膜或脑室下区所含神经干细胞较皮质更丰富 ,皮质来源的神经干细胞和室管膜来源的神经干细胞在形态学和分化程度上无特殊区别。该结果提示 ,SD胎鼠室管膜、皮质以及SD成年大鼠脑室下区、皮质 4种组织来源的细胞均含有神经干细胞 ;神经干?

To explore the feasibility of culture and identification of neural stem cells (NSCs) from ependyma, subventricular zone and cotex of fetal and adult rat respectively, and to make the basis for treatment of degenerative diseases with NSCs transplantation, all the seed cells derived from ependyma, subventricular zone and cotex were isolated from both fetal and adult SD rat, respectively. They were then cultured, induced to differentiation and subcultured continuously in a 'CYTOKINE NSCs culture medium'. Identification was carried out using Nestin, NSE and GFAP antibodies for differentiated NSCs, neuron and neuroglials, respectively. The seed cells from these four locations proliferated rapidly under some corresponding conditions, and formed 'neurologic spheres', which consisted of many cells and expressed Nestin antigen. After continuous culture and subculture, NSCs might divide and proliferate further. Some NSCs buds developed processes and formed nerve fibers further, while the soma enlarged into the cells with 'long processes', which connected or crisscrossed with each other, and were confirmed as neurons and neuroglias by immunocytochemistry. Seed cells from fetal rats might generate more NSCs than those from adult rats, and those from ependyma and subventricular zone produced more NSCs than those from cortex. There was no special morphological difference between ependyma NSCs and cortex NSCs. It is suggested that NSCs existed not only in ependyma and cotex of fetal SD rat, but also in the subventricular zone and cotex of adult SD rat. Fetal rat nerve tissue possesses much more NSCs than adult one.