大鼠脊髓损伤后细胞凋亡特点及其与一氧化氮合酶表达的相关性

目的探讨脊髓损伤后细胞凋亡特点与诱生型一氧化氮合酶(iNOS)表达规律及二者之间的关系.方法成年大鼠32只,随机分为8组(每组4只)单纯椎板切除对照组和脊髓损伤后7个时间点处死组(4、8 h和1、3、7、14、21 d).用免疫组化染色检测iNOS、p53阳性细胞,原位末端标记法(TUNEL)标记凋亡细胞.另取大鼠32只进行同样分组,用Western blot analsis法检测iNOS表达.结果免疫组化结果显示单纯椎板切除对照组神经元、神经胶质细胞、室管膜细胞、血管内皮细胞极少量表达iNOS;损伤后8 h上述细胞iNOS表达增加,7 d达高峰,14 d仍较明显,21 d降低.p53表达及TUNEL标记阳性细胞的时间分布特点与iNOS相似,7d达高峰,阳性细胞以白质中胶质细胞为主,主要分布于损伤部及相邻区.iNOS的Western blot灰度扫描数值时间变化曲线与免疫组化阳性细胞率时间变化曲线一致.iNOS表达与神经细胞凋亡指数及p53表达存在正相关(r＝0.854,P＜0.01;r＝0.951,P＜0.01).结论大鼠脊髓损伤后iNOS与p53表达增强,凋亡神经细胞大量出现.iNOS表达与脊髓损伤后神经细胞凋亡指数间存在正相关.

Correlation between apoptosis and expression of iNOS after spinal cord injury in rats

Objective To study the characteristic of apoptosis and expression of iNOS,as well as the relationship between them after spinal cord injury(SCI)in rats.Methods Thirty two adult SD rats were divided at random into eight groups:sham operated control group and acute SCI model groups that were sacrificed at 4,8 h,1,3,7,14 and 21 d after surgery.Then the sections were stained with immunohistochemistry and terminal deoxynucleotidy1 transferase mediated dUTP nick end labeling(TUNEL)method.Other 32 adult SD rats were divided at random into eight groups similarly and iNOS expression was detected in the injuried spinal cord tissue with Western blot analysis.Results Immunohistochemistry results demonstrated that a little iNOS immunoreactivity (iNOS IR) was detected in sham operated controls particularly in the regions surrounding the central canal and in the blood vessels.iNOS IR was distributed widely in gray matter neurons,white matter glial cells,ependymal cells and endothelial cells at the epicenter and adjacent to the region of spinal impact and injury,increasing at 8 hr with the most intense immunoreactivity observed in the white matter,around the central canal and in the blood vessels on day 7 post injury.The expression of p53 and the rates of TUNEL positive cells corresponded temporally to iNOS expression after SCI.The p53 immunoreactivity and TUNEL positive cells located mostly in white matter glial cells at day 7 post injury.The gray degree value time curve of iNOS expression detected by Western blot analysis corresponded to iNOS positive cells rates time curve.There was a positive correlation between iNOS expression and apoptosis index( r =0 854, P <0 01),as well as between iNOS expression and p 53 expression( r =0 951, P <0 01).Conclusions Expressions of iNOS and p53 are increased and a large number of TUNEL opsitive cells are present after SCI in rats.There is also a positive correlation between iNOS expression and apoptosis index.