大鼠继发性脊髓损伤中半胱氨酸蛋白酶水平的变化

Changes of Calpain levels in secondary spinal cord injury in rats

目的:通过半胱氨酸蛋白酶(Calpain)的活性变化探讨其在急性脊髓损伤中的作用。方法:将18只雌性SD大鼠随机分为Calpain高选择性抑制剂组(EST)、二甲基亚砜赋形剂对照组(DMSO)和空白对照组(Sham),每组6只。3组大鼠通过Allen′s方法(50gcm)制作急性脊髓损伤模型后,在15min内,EST组给予EST(1mg·kg-1),DMSO组给予1.5%DMSO,Sham组仅行椎板切除。24h后每组收集脊髓标本(长度约5.0cm),分成均等的5份:S1远头侧、S2近头侧、S3损伤段、S4近尾侧和S5远尾侧。通过Westernblotting对5份样品中的相对分子质量为68000神经丝蛋白(NFP)进行分析。结果:S1～S5段EST组和Sham组NFP条带密度值显著高于DMSO组(P<0.005),EST组与Sham组比较差异无显著性(P>0.05);Westernblotting条带分析S3段EST组NFP条带密度值较DMSO组高9.55,S4段高11.66(P<0.01)。结论:Calpain在急性脊髓损伤中活性显著增高,加重脊髓损伤。

Objective To observe the changes of activity of calpain in acute spinal cord injury with its highly selective inhibitor EST. Methods Methods:Eighteen S-D female rats were used to set up acute spinal cord injury models with Allen′s method. In 15 min, EST(1 mg·kg^-1) was administrated with intravenous injection in EST group,1.5% Climethyl sulfoxide (DMSO) was given in DMSO group, the rats in sham control group received nothing with laminectomy only. After 24 h, all rats were sacrificed and perfused via the left ventricle with 100 mL of phosphate buffered saline (PBS). Then a 5.0 cm long spinal cord section with the lesion in the center was collected from rat under anesthesia, and divided equally into 1.0 cm segments, S1 distant rostrol,S2 near rostrol,S3 lesion,S4 near caudal,S5 distant caudal, which were prepared for Western blotting analysis. Results The density values of neurofilament protein (NFP,68 000) band in EST group and sham group were significantly higher than that in DMSO group (P<0.005). EST could inhibit the degradation of NFP in various phases, the content of NFP increased significantly. Compared with DMSO group, the content of NFP in S3 section increased 9.55, while that in S4 section increased 11.66%(P<0.001). Conclusion Calpain activity is significantly enhanced in the rats following acute spinal cord injury.