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目的探讨不同胎龄胎鼠脊髓源性神经干细胞(NSCs)的增殖分化能力。方法 Sprague-Dawley孕鼠分为A组(孕12d)、B组(孕14 d)、C组(孕16 d),酶消化法结合机械分离法提取细胞,检测不同时间各组神经干细胞球的直径、数量,CCK-8法绘制细胞生长曲线。Brd U/Nestin免疫荧光染色鉴定NSCs。10%胎牛血清诱导分化后行β-tubulinⅢ、胶质纤维酸性蛋白(GFAP)免疫荧光染色,比较各组神经元的分化比例。结果各组细胞Brd U、Nestin、β-tubulinⅢ、GFAP免疫荧光染色均为阳性。B组β-tubulinⅢ染色阳性的比例最高;B、C两组各时间点神经球直径、数量及神经元的分化比例高于A组(P<0.05)。第3代NSCs培养第5天,B组细胞吸光度高于C组(P<0.05)。结论孕14 d左右取材培养的脊髓神经干细胞生物学特性稳定,增殖活力强,诱导分化后神经元的分化比例较高。
Objective To investigate the proliferation and differentiation of spinal cord-derived neural stem cells (NSCs) of different fetal mice. Methods Sprague-Dawley pregnant rats were divided into group A (pregnant 12 d), group B (pregnant 14 d), group C (pregnant 16 d), and enzyme digestion combined with mechanical separation to extract the cells. Diameter, number, CCK-8 method to draw cell growth curve. BrdU / Nestin immunofluorescence staining to identify NSCs. After induced by 10% fetal bovine serum, β-tubulinⅢ and glial fibrillary acidic protein (GFAP) were used for immunofluorescence staining. The differentiation ratio of neurons in each group was compared. Results The BrdU, Nestin, β-tubulin Ⅲ and GFAP immunofluorescence staining were positive in all groups. The ratio of β-tubulin Ⅲ staining in group B was the highest. The diameter and number of neurospheres and the proportion of neurons in groups B and C were higher than those in group A (P <0.05). On the fifth day after the third generation of NSCs cultured, the absorbance of cells in group B was higher than that in group C (P <0.05). Conclusion The neural stem cells cultured on the 14th day of gestation exhibited stable biological characteristics, strong proliferative activity and high differentiation rate of neurons after induced differentiation.