摘要
目的比较3个不同胚胎时期神经干细胞的增生、分化和凋亡等生物学性状,了解胚胎脑皮质发育过程。方法E14、E18和E20胎鼠前脑脑皮质来源的神经干细胞在原代培养后,用BrdU细胞增生检测试剂盒检测增生能力;诱导分化7天后,对细胞分别进行MAP2和GFAP免疫荧光标记和Hoechst33342核染色,计算MAP2+细胞和GFAP+细胞占Hoechst+细胞的各自比例;用AnnexinV-FITC凋亡试剂盒检测早期凋亡率。结果3组神经干细胞的OD值分别是:E14为1.1771±0.0422(n=22),E18为0.4127±0.0328(n=23),E20为0.2117±0.0456(n=16)(均为P<0.001);E14神经元细胞比例和星形胶质细胞比例分别为:81.96%±4.91%(n=10)和16.63%±4.05%(n=10),E18为27.56%±3.54%(n=10)和58.98%±5.91%(n=10),E20为4.75%±2.39%(n=10)和89.43%±3.40%(n=10)(均为P<0.001);早期凋亡率分别为:E14为41.12%±9.75%(n=8),E18为56.30%±9.35%(n=19),E20为5.74%±3.31%(n=9)(均为P<0.01)。结论神经干细胞的增生能力随着胚胎发育变得越来越弱,而凋亡比例先小后大,最后逐渐变小。神经干细胞分化为神经元细胞的比例随着胚胎发育过程会越来越小,而星形胶质细胞的比例会越来越大。
Objective With the comparison of the proliferation, differentiation and apoptosis of neural stem cells from different embryonic stages, to obtain the biology of NSCs and the development mechanism of embryonic cortex underlining. Methods After the prime cuhure of NSCs from forebrain cortice of different embryonic stages （ E14, E18 and E20）, enzyme immunoassay for NSCs proliferation was carried out. After 7 days of differentiation induced by fetal bovine serum, they were detected by the neuronal marker MAP2 and the astrocyte marker GFAP with nuclei staining by Hoechst 33342 additionally. The ratios of MAP2 ＋/Hoechst ＋ and GFAP ＋/Hoechst ＋ were calculated. Fluorescent activated ceU sorting for apoptosis were used to detect the apoptosis of NSCs by Annexin V - FITC and propidium iodide. Results The absorbanee of NSCs is 1. 1771 ±0.0422 （n =22） for El4, 0.4127 ±0.0328 （n =23） for El8, and 0.4127 ±0. 0456 （ n = 16） for E20 （P 〈 0. 001 ）. The ratios of MAP2 ＋/Hoeehst ＋ of the NSCs from El4, El8, and E20 were 81.96% ± 4.91% （n =10） , 27.56% ± 3.54% （n = 10） , and 4.75% ± 2.39% （n = 10） respectively, while the ones of GFAP ＋/Hoeehst ＋ were 16. 63% ＋4.05% （n=10）, 58.98% ±5.91% （n=10）, and 89.43% ±3.40% （n=10）（P〈0.001）. Analyses ofapoptosis using eytoflurometry are 41.12% ±9.75% for El4 （n =8）, 56.30%±9.35% for El8 （n =19）, and 5.74% ±3.31% （n =9） （P〈 0.01 ）. Conclusion The proliferation of NSCs decreases during the development of embryonic brain while the apoptosis takes on a fluctuation, which increases before decreasing. During the development of embryonic brain, NSCs differentiate into less neurons and more astroeytes.
出处
《医学研究杂志》
2008年第5期19-22,F0003,共5页
Journal of Medical Research
基金
浙江省自然科学基金资助项目(RC02062)
关键词
神经干细胞
增生
分化
凋亡
Neural stem cell
Proliferation
Differentiation
Apoptosis
