神经干细胞提取与培养方法的比较研究

Comparative study on extraction and culture method of neural stem cells

目的探讨神经干细胞(neural stem cells,NSCs)提取、培养的最佳方法,为NSCs的基础研究提供技术参考。方法对NSCs的不同提取、培养方法进行比较:提取胎鼠与乳鼠的大脑皮质、采用不同类型培养基、不同接种密度、不同培养方法、不同换液方法、不同传代方法,观察NSCs的成球率和NSCs未分化状态的稳定性。用多功能微孔板读数仪检测NSCs活性。结果 (1)胎龄14 d胎鼠较新生24 h内乳鼠的大脑皮质提取NSCs的比例高,杂细胞少,形成的神经球数量多。(2)采用无血清培养基,NSCs可以稳定在未分化状态;而采用含血清培养基,NSCs多数分化为神经元和胶质细胞。(3)以1.0×109·L^(-1)密度接种的NSCs,培养形成的神经球数量多,且状态良好。(4)悬浮培养法较贴壁培养法,可以形成稳定的神经球,有利于保持NSCs处于未分化的状态。(5)半量换液法和只加液不弃液法培养形成的神经球的状态优于全量换液法。(6)原代和1代的神经球用机械吹打法传代后,NSCs的活细胞比例高,再次形成的神经球状态好,且较其他两种方法简便;2代及以后的神经球用Accutase酶传代后,NSCs的活细胞比例高,再次形成的神经球状态好。(7)14 d胎鼠Accutase酶消化组的NSCs活性高于另外3组,差异均具有显著性(P<0.05)。结论提取胎龄14 d胎鼠的大脑皮质,采用无血清培养基,以1.0×10~9·L^(-1)1的密度接种于25 cm^2培养瓶,采取悬浮培养法,每2~3 d加液1~1.5m L,每6~7 d传代1次,所得到的NSCs增殖分裂旺盛,成球率高,神经球状态良好,可以保持稳定的未分化状态。

Aim To explore the best method of neural stem cell extraction and culture,and provide a technical reference for the basic research of neural stem cells.Methods Different extraction and culture methods of neural stem cells were compared.The rate of ball of neural stem cells and the stability of neural stem cells in undifferentiated state were observed by extraction of fetal and neonatal rats cortex,using different types of medium,different inoculation density,different culture methods,different methods of changing liquid and different passage methods.Neural stem cells’activities were detected by Varioskan LUX Multimode Microplate Reader.Results ①The brain cortex of fetal rats of 14 d had higher proportion of neural stem cells,less other cells and more neurospheres than newborn rats of 24 h.②Neural stem cells could be stabilized in undifferentiated state by using serum-free medium,while most of the neural stem cells were differentiated into neurons and glial cells by using serum medium.③Neural stem cells,seeding at 1.0×10 9·L-1,had a large number of neurosperes and were in good condition.④Suspension culture was beneficial to form a stable neurosphere and keep the neural stem cells in an undifferentiated state than adherent culture.⑤The state of neurosphere by changing half of the medium and adding medium without discarding was better than that by replacing all medium.⑥The neurospheres could be separated into single cells by mechanical blow in primary generation and the second generation of neurospheres cultured in serum-free medium.While the percentage of viable cells in neural stem cells was the highest digested with stem cell lysates after three generations and the neurospheres re-formed were better.⑦Neural stem cells’activity of 14 d fetal rat in Accutase digestion group was significantly higher than that of the other three groups,and the difference was significant(P<0.05).Conclusions Neural stem cells proliferate and divide well,with high rate of ball formation and good neurosphere condition,which can maintain a stable undifferentiated state by extracting the cerebral cortex of 14 d fetal rats,using serum-free medium,inoculating into a 25 cm 2 flask at a density of 1.0×10 9·L-1,and taking the suspension culture(adding the medium 1~1.5 mL every 2~3 d and passage every 6~8 d).