Objective To compare the proliferation of neural stem cells ( NSCs ) with two different culture methods. Methods Primary NSCs isolated from whole brain of neonatal Sprague Dawley rats ( 24h after birth ) were suspended and cultured in serum - free medium for long - term survival in vitro. The cells were passaged continuously with either mechanical dissociation or trypsinization method. Immunocytochemistry was applied to detect the expression of neuroepithelial stem cell protein ( Nestin ), neuron specific enolase ( NSE ) , glial fibrillary acidic protein ( CFAP ) and cyclic nucleotide phosphohydrolase ( CNP ). Results The cells isolated from neonatal SD rats possessed the ability to proliferate and self - renew, with nestin - positive neurospheres, and multi - differential ability. Either in P1 or P2, significantly more NSCs were detected in the trypsinization group than those in the mechanical dissociation group. Conclusion NSCs are successfully isolated from SD rats with self - renewing, proliferating and differential abilities. Trypsinization method possesses higher efficacy in NSCs culture.%目的 比较两种不同的分离培养方法对大鼠神经干细胞(NSCs)增殖的影响.方法 分离新生24 h内的SD大鼠脑组织细胞,采用无血清培养技术进行体外扩增培养,分别以机械吹打法和胰酶消化法两种方法进行扩增培养、传代.免疫细胞化学法对NSCs 及其分化后的细胞进行Nestin、NSE、GFAP、CNP的鉴定.结果 分离培养的细胞具有自我更新和增殖能力及Nestin 表达阳性,诱导后可分化为神经元、星形胶质细胞及少突胶质细胞.无论第1代或第2代比较,胰酶消化组NSCs 的增殖明显高于机械吹打组.结论 实验中分离培养的细胞为具有自我更新和增殖能力的NSCs,可诱导分化为终末神经细胞.在两种分离培养方法中,以胰酶消化法培养可获得更多的NSCs.
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