G-CSF对大鼠脊髓损伤后神经细胞凋亡及Bcl-2、Bax蛋白表达的影响

目的 通过观察粒细胞集落刺激因子（G-CSF）对大鼠急性脊髓损伤后神经细胞凋亡及Bcl-2、Bax蛋白表达的影响,探讨其对脊髓损伤的保护机制.方法 32只Wistar大鼠采用改良的Alien＇s装置在T11水平制成大鼠脊髓损伤模型,随机分成2组：对照组和G-CSF治疗组.在术前及术后各时间点对大鼠进行Basso-Beattie-Bresnahan（BBB）评分评价大鼠后肢神经功能恢复情况;用免疫荧光法检测脊髓损伤后各时间点Bcl-2、Bax蛋白表达;Tunel法检测凋亡细胞数量.结果 大鼠脊髓损伤后3d,在脊髓损伤部位可见大量的TUNEL阳性细胞,7d时达到高峰,此后表达量逐渐下降,21d时仍可见少量阳性细胞;损伤后3d时即出现大量Bcl-2和Bax阳性细胞,伤后7d时达高峰,21d时仍有少量表达.与对照组相比,治疗组3、7和14d时Bcl-2蛋白表达增高及Bax蛋白表达减少有统计学意义（P〈0.01）;治疗组3、7、14（P〈0.01）和21d（P〈0.05）时凋亡细胞数减少有统计学意义;BBB评分显示大鼠神经功能恢复显著优于对照组.结论 G-CSF可以减少大鼠脊髓损伤后的神经细胞凋亡,从而发挥神经保护作用;作用可能是通过上调Bcl-2和下调Bax蛋白的表达实现.

The effect of granulocyte colony-stimulating factor on changes of neuronal apoptosis and the protein expression of B-cell lymphoma-2 and Bcl-2-associated X protein after spinal cord injury in rats

Objective To investigate the protective mechanism of granulocyte colony-stimulating factor （G-CSF） by observing the effect of G-CSF on changes of neuronal apoptosis as well as the protein expression of B-cell lymphoma-2 （Bcl-2） and of Bcl-2-associated X （Bax） after spinal cord injury in rats. Methods A total of 32 adult Wistar rats were made into the models of spinal cord injury at T11 level according to the improved Allen＇s device. They were randomized into 2 groups, group A as control, and group B treated with G-CSF. At each time point pre-operatively and post-operatively, the rats were evaluated according to the Basso-Beattie-Bresnahan Locomotor Rating Scale （BBB） in that the recovered level of neurological function of posterior limb in rats were assessed. The protein expression of Bcl-2 and Bax was detected according to the immunofluorescence method after the spinal cord injury at each time point. The apoptosis was qualitatively assessed according to the Tunel method. Results 3 days after the spinal cord injury, the rats reported plenty of positive Tunel cells in the lesion. In 7 days, positive Tunel cells reached the maximum peak in quantity, and in 21 days, a small amount of them still visibly existed. Of positive cells of Bcl-2 and Bax, a large quantity of them were detected post-operatively in 3 days, the amount reached the peak in 7 days, and a few were still expressed in 21 days. In comparison to group A, group B was significantly different in the increase of Bcl-2 protein expression and in the decrease of Bax protein expression in 3 days, 7 days, and 14 days （P〈0.01）. Of the reduction of apoptosis amount, significant difference was found between group A and group B in 3 days, 7 days, and 14 days （P〈0.01） as well as in 21 days （P〈0.05）. The recovered level of neurological function in rats in group B was significantly better than group A. Conclusions G-CSF can protect the nerve through the reduction of the neuronal cell apoptosis in rats after spinal cord injury, which may be due to the up-regulation of the Bcl-2 protein expression, and down-regulation of the Bax protein expression.