新生大鼠神经干细胞的体外培养方法

摘要

Objective To investigate an appropriate method for culturing neural stem cells ( NSCs ) from newborn SD rats. Methods NSCs were isolated from the brain of newborn SD rats 1-3 days after birth. NSCs were cultured with different seeding density and different passage methods. The growth of NSCs was observed. NSCs were identified by detecting the expression of Nesting neuron specific enolase ( NSE ) and glial fibrillary acidic protein ( GFAP ) with immunocytochemistry. Results The neurospheres were Nestin - positive and could differentiate into NSE - positive neurons and GFAP -positive glial cells. The cultured cells with the ability of self - renewal and pluripotent differentiation were proved to be NSEs. When the seeding density was 1 × 106/mL,NSCs proliferated quickly and generated more neurospheres. The method of passage should be selected according to different purposes. The time of passage was about 7 days. Conclusion The established method is simple and stable for culturing NSCs from newborn SD rats in vitro.%目的 探讨新生sD大鼠神经干细胞(neural stem cells,NSCs)适宜的体外分离培养方法.方法 从出生1～3d的SD大鼠脑组织中分离NSCs,采用不同的接种密度、传代方法,在体外培养NSCs,观察细胞的生长情况.巢蛋白(Nestin)、神经元特异性烯醇化酶(neuron specific enolase,NSE)、胶质原纤维酸性蛋白(glial fibrillary acidic protein,GFAP)免疫细胞化学法对NSCs进行鉴定.结果 培养的神经球Nestin表达阳性,血清诱导后可分化为NSE阳性神经元和GFAP阳性星形胶质细胞,具有自我增殖和多向分化能力,是NSCs.当NSCs接种密度为1×106/mL时,细胞增殖速度快,神经球生成的数量多.传代的方法应视情况而定,传代时间为7d左右.结论 建立了简单、稳定的新生大鼠NSCs的体外培养方法,为进一步研究NSCs的生物学特征及组织移植奠定了基础.