神经营养因子复合剂N6对脊髓损伤神经细胞凋亡的作用

Effect of a neurotrophic factor compound(N6) on the cell apoptosis secondary to spinal cord injury

[目的]研究神经营养因子复合剂(N6)对脊髓损伤后神经细胞凋亡的作用。[方法] Wistar大鼠(SPF级) 48只随机分为4组,每组12只。假手术组,只摘除椎板未损伤脊髓;单纯脊髓损伤组(损伤组),采用Allen法经NYU脊髓损伤撞击器制备急性脊髓损伤模型;脊髓损伤+生理盐水治疗组(INS组),制备急性脊髓损伤模型,并给予蛛网膜下腔注射生理盐水治疗;脊髓损伤+N6治疗组(IN6组),制备急性脊髓损伤模型,并给予蛛网膜下腔注射N6治疗。损伤后72 h取材,采用HE染色对脊髓组织进行标记;免疫组化方法检测Caspase 3蛋白的表达;半定量逆转录-聚合酶链式反应(RT-PCR)测定凋亡相关基因Caspase 3、Fas、Bax、Bcl2、P53 mRNA在脊髓组织中的表达变化。[结果]免疫组化结果显示:假手术组脊髓神经细胞很少有Caspase 3表达,假手术组较损伤组和INS组的Caspase 3阳性表达细胞数明显减少(P<0.01);与之类似,IN6组较损伤组和INS组Caspase-3阳性表达细胞数也明显减少(P<0.01)。RT-PCR检测到Caspase-3、Fas和P53 mRNA水平,IN6组较损伤组和INS组明显降低(P<0.05);而Bcl2/Bax值,IN6组较损伤组和INS组明显升高(P<0.05)。[结论]神经营养因子复合剂N6能明显抑制脊髓损伤后神经细胞的凋亡。

[Objective] To explore the effect of a neurotrophic factor compound(N6) on apoptosis of nerve cells after acute spinal cord injury. [Methods] Forty-eight Wistar rats were used in this study, and divided randomly into 4 groups with 12 animals in each group. The animals in the sham-operated group(the sham group) had only laminectomy conducted without spinal cord injury created, those in the injured group got acute spinal cord injury(SCI) with the NYU impactor by the Allen.s method without any following treatment, while those in the injured plus normal saline treatment group(the INS group) received subarachnoid injection of normal saline after SCI created, and the animals in the injured plus N6 treatment group( the IN6 group) received subarachnoid injection of N6 after SCI created. The spinal cord tissues of the lesion epicenter were harvested at 72 hours after injury. The hematoxylin and eosin(HE) staining for histopathological changes, immunohistochemisty for Caspase 3 protein expression, and reverse transcription polymerase chain reaction(RT-PCR) for expression of apoptosis-related genes, including Caspase 3, Fas, Bax, Bcl2 and P53, were conducted for assessments. [Result] The immunohistochemisty revealed that expressed Caspase 3 in the sham group was significantly less than the injured group and INS group(P<0.01), in the same way, the expressed Caspase 3 in the IN6 group was also significantly less than the injured group and INS group(P<0.01). Regarding to RT-PCRs, the mRNA expression of caspase-3, Fas and P53 in the IN6 group were significantly lower than the injured groupPBcl2/Bax proved significantly higher in the IN6 group thanthe injured group and INS group(P<0.05). [Conclusion]The neurotrophic factor compound(N6) does obviously inhib-it the apoptosis of nerve cells following acute spinal cord in-jury.