神经生长因子对神经干细胞增殖、分化和凋亡的影响

目的 观察不同剂量神经生长因子(NGF)对神经干细胞增殖、分化和凋亡的影响.方法 从孕15 d鼠胚胎脑室下区组织分离神经干细胞,培养于DMEM/F12+B27细胞基础培养液中,加入碱性成纤维细胞生长因子(bFGF)作为阳性对照组,实验组分别加入10、20、50μg/L的NGF,无添加成分作为阴性对照组.用噻唑蓝(MTT)比色分析法测定细胞增殖能力;倒置显微镜下观察细胞增殖、分化,鼠抗胶质纤维酸性蛋白(GFAP)、鼠抗神经元特异性烯醇化酶(NSE)免疫细胞化学染色鉴定;流式细胞仪检测细胞凋亡率.结果 10 d MTT比色显示,10μg/L NGF组(0.150±0.025)与阳性对照组(0.158±0.017)比较,差异无统计学意义(P＞0.05),与阴性对照组(0.128±0.015)比较,差异有统计学意义(P＜0.05).分化实验显示:随着NGF的浓度递增,NSE阳性率分别为(15.21±1.27)%、(20.64±2.61)%、(24.20±3.10)%,GFAP阳性率分别为(27.98±2.35)%、(28.94±2.84)%、(30.79±3.23)%,神经干细胞分化的比率增高FNsz(+)=3.47、FGFAP(+)=3.47,PGFAP(+)＜0.05].流式细胞仪检测示20、50μg/L NGF组细胞凋亡率明显增高,l0μg/L NGF组细胞增殖指数(PI)较其他实验组高.结论 合适浓度NGF能促进神经干细胞的增殖.随神经生长因子浓度的增高,促神经分化作用增强.随着NGF浓度的增加,其促NSCs凋亡作用增强.

Abstract：

Objective To evaluate the influence of different doses of nerve growth factor (NGF) on the proliferation, differentiation and apoptosis of embryonic rat neural stem cells. Methods The neural stem cells were isolated from subventricular zone of rat embryonic brains and cultured in serum-free medium DMEM/F12 with B27. NGF with different doses of 10, 20, 50 μg/L were added into the different experimental groups. The proliferation ability of the cells was measured by methyl thiazol tetrazolium (MTT) assay at 10th day. All cells were collected in plates at 7th day. Streptvidin-peroxidase immunocytochemistry was used to detect the expression of neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP) , and the number of NSE( + ) and GFAP( + ) cells was counted. The apoptosis rat at 5th day after culture was assessed. Results There was no significant difference in the the number of neurospheres between 10 μg/L NGF group (0. 150 ±0. 025) and positive control group (0. 158 ±0. 017), but there was significant difference between 10 μg/L NGF group and blank control group (0. 128 ±0. 015). In 10, 20,50 μg/L N GF groups, the rate of NSE ( + ) cells was ( 15. 21 ± 1.27) %, (20. 64 ± 2. 61 )%, (24. 20 ± 3. 10) %, and that of GFAP( + ) cells was (27.98 ± 2. 35 ) %, ( 28.94 ± 2. 84 ) %, ( 30. 79 ± 3.23 ) % respectively. The rate of cells differentiation was increased with the dose of NGF added( FNsE (+ ) = 3.47,FGFAp( + ) = 3. 47 ,PGFAP( + ) ＜ 0. 05)]. Flow cytometry revealed that in 20, 50 μg/L NGF groups the apoptosis rate was significantly increased as compared with other groups. The proliferation index (PI) in 10 μg/L NGF group was higher than other groups. Conclusion NGF at a rational dose can promote the proliferation and differentiation of human NSC. Low dose of NGF mainly facilitates the proliferation of NSCs, and high dose of NGF shows obvious influence on proliferation of NSC.

Influence of nerve growth factor on proliferation, differentiation and apoptosis of neural stem cells

Objective To evaluate the influence of different doses of nerve growth factor (NGF) on the proliferation, differentiation and apoptosis of embryonic rat neural stem cells. Methods The neural stem cells were isolated from subventricular zone of rat embryonic brains and cultured in serum-free medium DMEM/F12 with B27. NGF with different doses of 10, 20, 50 μg/L were added into the different experimental groups. The proliferation ability of the cells was measured by methyl thiazol tetrazolium (MTT) assay at 10th day. All cells were collected in plates at 7th day. Streptvidin-peroxidase immunocytochemistry was used to detect the expression of neuron specific enolase (NSE) and glial fibrillary acidic protein (GFAP) , and the number of NSE( + ) and GFAP( + ) cells was counted. The apoptosis rat at 5th day after culture was assessed. Results There was no significant difference in the the number of neurospheres between 10 μg/L NGF group (0. 150 ±0. 025) and positive control group (0. 158 ±0. 017), but there was significant difference between 10 μg/L NGF group and blank control group (0. 128 ±0. 015). In 10, 20,50 μg/L N GF groups, the rate of NSE ( + ) cells was ( 15. 21 ± 1.27) %, (20. 64 ± 2. 61 )%, (24. 20 ± 3. 10) %, and that of GFAP( + ) cells was (27.98 ± 2. 35 ) %, ( 28.94 ± 2. 84 ) %, ( 30. 79 ± 3.23 ) % respectively. The rate of cells differentiation was increased with the dose of NGF added( FNsE (+ ) = 3.47,FGFAp( + ) = 3. 47 ,PGFAP( + ) ＜ 0. 05)]. Flow cytometry revealed that in 20, 50 μg/L NGF groups the apoptosis rate was significantly increased as compared with other groups. The proliferation index (PI) in 10 μg/L NGF group was higher than other groups. Conclusion NGF at a rational dose can promote the proliferation and differentiation of human NSC. Low dose of NGF mainly facilitates the proliferation of NSCs, and high dose of NGF shows obvious influence on proliferation of NSC.