Objective To observe the protective effects of basic fibroblast growth factor (bFGF) on injured neurons by establishing injured neuron model in vitro in order to understand the effect of bFGF on neurons and its mechanism and to provide new treatment for neuron injury. Methods The glutamate (50 μmol/ L) was added into the 10-day primarily cultured hippocampal neurons, and then the morphologic changes of cells was observed through contrast phase microscope. The neuron survival rate was tested by using trypan blue staining method and the neuron apoptosis rate was tested by TUNEL technique to evaluate the degree of injury on hippocampal neurons by glutamate. The low concentration glutamate was used to induce neuron apoptosis. Immunocytochemistry techniques, trypan blue staining method and TUNEL technique were also used. bFGF (20 ng/ml) was added into the experimental group. The protective effects of bFGF on the survival of injured neuron and the caspase-3 expression in injured neurons were observed. Results After hippocampal neurons were induced by glutamate for 4 hours, part of the cells became round and swelling with the decurtation or deprivation of the apophysis. 24 hours later, most of the swollen cells disintegrated. Trypan blue staining method was used to test the neuron survival rate. The neuron survival rate in experimental group (83. 8 ± 6. 8) % showed statistically significant difference compared with that in the control group (77. 4 ± 6. 5) % (t = 8. 52, P <0. 05). After hippocampal neurons were induced by glutamate for 24 hours, TUNEL staining showed the shrinkage of the cell surface, the diminution of volume and pycnosis. The apoptosis rate of the neurons cells in experimental group (1.5 ±0. 7) % showed statistically significant difference compared with that in the control group (2. 7 ± 1. 4) % (t =-10. 35 , P <0. 05). The differences of the survival rate in the three groups three days within apoptosis all showed statistical significances (P<0. 05).rnAmong them, the survival rate in normal group increased significantly compared with the experimental group and the control group. The experimental group also increased significantly compared with the control group. The differences all have statistical significance (P <0. 05). The apoptosis rate of the three groups within three days after apoptosis all showed statistically significant differences (P <0. 05). Among them, the apoptosis rate in normal group decreased significantly compared with the experimental group and the control group. The experimental group also decreased significantly compared with the control group (P<0. 05). The positive expression rate of caspase-3 in the three groups within three days after apoptosis all showed statistically significant differences (P <0. 05). The positive expression rate of caspase -3 in normal group decreased significantly compared with the experimental group and the control group. The positive expression in experimental group also decreased significantly compared with the control group (P <0. 05). Conclusion After bFGF was added into hippocampal neuron, the neuron survival rate will increase but the neuron apoptosis rate and caspase-3 positive expression rate will decrease, indicating the bFGF can protect the impaired neuron. bFGF may play its protective role through decreasing the activation of caspase-3 pathway and inhibiting cell apoptosis.%目的 建立体外神经元损伤模型,观察碱性成纤维细胞生长因子(bFGF)对损伤神经元存活的保护作用,了解bFGF对神经元的作用和机制,为神经损伤提供新的治疗措施.方法 在体外培养10 d的海马神经元中加入50 μmol/L的谷氨酸,相差显微镜下观察细胞形态变化,用苔盼蓝染色法检测存活率和细胞凋亡原位(TUNEL)检测凋亡率,来评定谷氨酸对海马神经元的损伤程度.以低浓度谷氨酸诱导神经元凋亡,应用免疫组化、苔盼蓝染色法和TUNEL检测等技术,在实验组中加入bFGF(20 ng/ml),观察bFGF对损伤神经元存活的保护作用以及caspase-3基因在损伤神经元中的表达情况.结果 谷氨酸作用于海马神经元4 h后,可见部分细胞变圆,胞体肿胀,突起缩短或消失;24 h后大部分肿胀细胞崩解.通过苔盼蓝染色法检测神经元存活率,实验组神经元存活率[(83.8±6.8)%]与对照组[(77.4±6.5)%]比较,差异有统计学意义(t=8.52,P＜0.05);谷氨酸作用于海马神经元24 h后,TUNEL检测显示凋亡神经元表面皱缩,体积缩小,胞核固缩.实验组神经元凋亡率[(1.5±0.7)%]与对照组[(2.7±1.4)%]比较,差异有统计学意义(t=-10.35,P＜0.05).凋亡后3 d内3组的神经元存活率比较,差异均有统计学意义(P＜0.05);其中正常组较实验组和对照组神经元存活率均显著增加,实验组较对照组亦显著增加,差异均有统计学意义(P＜0.05).凋亡后3 d内3组的神经元凋亡率比较,差异均有统计学意义(P＜0.05),其中正常组较实验组和对照组神经元凋亡率均显著减少,实验组较对照组亦显著减少,差异均有统计学意义(P＜0.05).凋亡后3 d内3组caspase-3基因阳性表达率比较,差异均有统计学意义(P＜0.05),正常组较实验组和对照组caspase-3基因阳性表达率均显著减少,实验组较对照组亦显著减少,差异均有统计学意义(P＜0.05).结论 在神经元中加入bFGF后,神经元存活率升高,神经元凋亡率和caspase-3基因阳性表达率降低,提示bFGF对损伤神经元有保护作用.bFGF可能是通过减少caspase-3通路激活,进而抑制神经元凋亡,从而发挥对神经元的保护作用.
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