不同营养因子组合对大鼠脊髓神经元的作用研究

Combined effects of different neurotrophins on rat spinal cord neurons

[目的]探讨NGF、bFGF和BDNF的不同组合对大鼠脊髓神经元的作用。[方法]利用神经元培养技术,将新生1 d的SD大鼠脊髓神经元接种于培养板,根据不同因子两两组合分为3个实验组、空白对照组和单因子对照组,因子终浓度均为50 ng/ml。倒置相差显微镜动态观察,接种后第3、7 d细胞爬片行β-tubulin3免疫荧光染色和Hoechst染色。测量阳性细胞最长突起长度,计算阳性细胞率,第1、3、5、7、9 d行MTT检查,以OD值绘制生长曲线。[结果]各实验组神经元突起长度均优于对照组(P<0.05),联合应用组优于单因子组,其中以A组(BDNF+bFGF)突起最长,各实验组β-tubulin3阳性细胞率均高于对照组,MTT结果与染色结果一致。[结论]特定浓度下NGF、bFGF及BDNF可以有效促进大鼠脊髓神经元轴突的生长,并可有效维持神经元细胞存活,联合应用作用增强,BDNF+bFGF作用最强。

[ Objective] To investigate the combined effects of different neurotrophin couples of NGF, bFGF, BDNF on rat spinal cord neurons. [ Method] Spinal cord neurons were obtained from SD rats born within ld, and then were seeded in culture plates. Different factors and their couples were added in each chamber while DMEM/F12 served as controls, concentration of NGF, bFGF or BDNF were 50ng/ml. Phase contrast microscope observation was done. At the 3^rd and 7^th day after incubation, the cells were detected by β-tubulin3 immunofluorescence and Hoechst staining. The length of neuritis was measured, and the numbers of neuron cells and nuclears were determined. At the 1^st, 3^rd, 5^th, 7^th and 9^th day MTT method was used, and the growth curve was made according to OD results. [ Result] The length of axon and positive rate in the experimental groups were superior to those in the control group （ P 〈 0.05 ）. The effects in the combined groups were superior to the single groups, especially in the group of BDNF ＋ bFGF. MTT method produced the same results as staining. [ Conclusion] NGF, bFGF and BDNF can enhance rat spinal cord neuronal neurite outgrowth and survival. Combined use of any two can produce better results, especially for the combined use of BDNF and bFGF.