大鼠牵张性脊髓损伤后细胞凋亡及相关基因表达的实验研究

An experimental study of cell apoptosis and correlative gene expression after tractive spinal cord injury in rats

目的观察大鼠牵张性脊髓损伤后细胞凋亡现象,检测脊髓损伤后凋亡相关基因的表达。方法大鼠脊髓T13～L2经牵张损伤,皮层体感诱发电位(CSEP)监测P1N1波幅下降至术前波幅70%后,分别于术后30min、6h、1、4、7、14、21d处死大鼠,取材(n=4)。应用流式细胞仪、原位末端脱氧核糖核苷酸转移酶介导dUTP标记(TUNEL)技术观察脊髓细胞凋亡情况,用免疫组化检测p53、bax和bcl2的表达。结果流式细胞仪及TUNEL法检测显示,损伤组术后6h凋亡细胞开始增多,术后7d细胞凋亡率达高峰,随后开始回落,持续21d,与空白对照组及椎板切除组比较,差异有显著性意义(P<005,001)。TUNEL法染色显示,白质中出现大量胶质细胞凋亡。免疫组化染色显示损伤组术后6h开始,p53、bax和bcl2阳性表达开始增多,p53阳性细胞数术后4d达高峰,bax和bcl2术后7d达高峰,损伤组各时相点的阳性表达与空白对照组与椎板切除组比较差异有显著性意义(P<005,001)。结论牵张性脊髓损伤后存在细胞凋亡现象,从形态上看包括神经元和胶质细胞凋亡,细胞凋亡是牵张性脊髓损伤继发损害中细胞死亡的一种重要形式,也是继发损伤期的重要病理变化。凋亡相关基因p53、bax大量表达,可能在脊髓细胞凋亡过程中起重要作用。

Objective To observe the cell apoptosis after tractive spinal cord injury in rats, determine expression of apoptosic correlative genes, and study the molecular mechanism of cell apoptosis. Methods The T 13 L 2 spinal cord of rats was injured by traction after the amplitude of P1 N1 wave decreased to 70% in postoperation than in preoperation through cortical somatosensory evoked potential (CSEP) monitor. Then rats were killed in 30 min、6 h、1、4、7、14 and 21 d respectively after operation ( n =4) .Cell apoptosis was examined by the flow cytometer and terminal deoxynucleotidal transferase mediated DUTP biotin nick end labeling (TUNEL) reaction, the expression of p53, bax and bc1 2 genes was tested with immunohistochemistry. Results The flow cytometer test and TUNEL method showed that the apoptosic cell ratio raised in 6 h and reached at peak in 7 d after injury, and then declined till 21 d, they showed significant difference ( P <0 05,0 01).TUNEL method showed that injured group had a large number of apoptosic glial cells in white matter. Immunohistochemical staining showed that the positive expression of p53、bax and bc1 2 protein raised at 6 h, expression of p53 protein reached at peak in 4 d, bax and bc1 2 protein reached at peak in 7 d after injury. Compared with control group and laminectomy group, the injuried group showed significant difference ( P <0 05,0 01). Conclusion There is cell apoptosis phenomenon after tractive spinal cord injury in ratis. Morphology indicates that apoptosis includes neurons and glialcytes, which is an important form of cell death and pathological changes in secondary lesion period after tractive spinal cord. There exist high expression of apoptosic correlative gene p53 and bax after spinal cord injury, they may play an important role in reduction of cells to apoptosis.