机械吹打法、胰酶消化法分离新生大鼠脑组织神经干细胞效果观察

Effects of mechanical isolation and pancreatin digestant isolation in separating neural stem cells from brain tissues of neonatal rats

目的对比观察机械吹打法、胰酶消化法分离培养新生大鼠脑组织神经干细胞(NSCs)的效果。方法取出生3天的新生SD大鼠大脑皮质,分别用机械吹打法、胰酶消化法分离培养(分别记为机械吹打组和胰酶消化组),通过免疫荧光法对NSCs进行鉴定和诱导分化;分别于培养第2、5、7天计数细胞球直径及数目;通过MTT法检测两组原代及第3亚代细胞的克隆增殖能力;选取第3亚代细胞进行冻存、复苏,计算细胞复苏后的存活率。结果两组均可从新生SD大鼠大脑皮质分离得到NSCs,并均可诱导分化为神经元、神经胶质细胞。培养5、7天时,机械吹打组获得的细胞球数量和直径均高于相同培养时间的胰酶消化组(P均<0.05)。机械吹打组原代细胞培养2、5、7天细胞克隆增殖能力均高于相同培养时间的胰酶消化组(P均<0.05),第3亚代细胞培养2、5天细胞克隆增殖能力均优于相同培养时间的胰酶消化组(P均<0.05)。两组第3亚代细胞冻存、复苏后存活率比较差异无统计学意义(P>0.05)。结论机械吹打法、胰酶消化法均可成功分离培养、诱导分化新生大鼠NSCs,机械吹打法分离培养的NSCs克隆增殖能力高于胰酶消化法。

Objective To compare the effects of mechanical isolation and pancreatin digestant isolation in separating neural stem cells（ NSCs） from brain tissues of neonatal rats. Methods The cerebral cortex of newborn rats within 3 days were isolated and cultured by mechanical and pancreatin digestion,respectively. The NSCs were identified and differentiated by immunofluorescence assay. Diameter and number of cell spheres were detected on day 2,5 and 7 of culture. The proliferation ability of the primary passage of NSCs and the third-passage NSCs in the two groups were compared by MTT.The third-passage NSCs were frozen and recovered and the survival rate of the cells was calculated. Results Both of these two methods could isolate NSCs from the cerebral cortex of neonatal rats,and those NSCs could be induced to differentiate into neurons and glial cells. On day 5 and 7,the NSCs＇ ball number and diameter which came from the mechanical isolation group were higher than those of the pancreatin digestant group in the same culture time（ all P 〈0. 05）. On day 2,5and 7,the prime NSCs which came from the mechanical isolation group had better clonal proliferation ability than those of the pancreatin digestant group in the same culture time（ all P〈 0. 05）. On day 2 and 5,the third-passage NSCs of the mechanical isolation group had better clonal proliferation ability than those of the pancreatin digestant group in the same culture time（ all P 〈0. 05）. The survival rates of the third-passage NSCs of the mechanical isolation group and pancreatin digestant group were respectively 85% and 89%,respectively; and there was no significant difference between these two groups（ P 〉0. 05）. Conclusion Both mechanical isolation and pancreatin digestant isolation can successfully isolate NSCs.NSCs which come from the mechanical isolation group have better clonal proliferation ability than those of the pancreatin digestant group.