MCAO大鼠不同时相凝血酶mRNA、凝血酶蛋白的变化规律探讨

Changes in mRNA and protein of thrombin at different time phases in rats with middle cerebral artery occlusion

目的：通过观察大脑中动脉闭塞（MCAO）大鼠模型不同时相神经功能缺失评分以及脑组织凝血酶mRNA和蛋白的表达,探讨MCAO大鼠缺血后神经功能缺损症状及凝血酶的变化规律,为药物研究确定最佳观察时间点。方法：雄性SD大鼠35只,随机分为假手术组和缺血后不同时相（1、6、12、24、48、72h）组,每组5只。对缺血后不同时相组采用线栓法制造MCAO大鼠模型,各组在取材前均进行神经功能缺失评分,用荧光定量实时PCR法检测大鼠脑组织凝血酶mRNA的表达水平;Western blot法检测大鼠脑组织凝血酶蛋白表达水平。结果：假手术组大鼠无神经功能损伤的症状,缺血后1~72h组大鼠表现为对侧前爪内收、向对侧转圈行走、向对侧倾倒等,以缺血后24h最为显著;大鼠脑缺血后1h凝血酶基因表达即增强且于24h达峰值;凝血酶蛋白的表达在脑缺血后1h即增多,持续上升到24h,然后维持在较稳定的高水平。结论：MCAO大鼠在急性脑缺血后凝血酶mRNA、凝血酶蛋白表达均增强,其峰值均集中在缺血后24h,此时大鼠神经功能缺损症状也最为显著,提示凝血酶可能参与了急性脑缺血的病理损害,缺血后24h可作为药物研究的最佳观察时间点。

Objective： To investigate the symptoms of neurologic impairment and changes in thrombin by observing neurologic impairment scores and the mRNA and protein expression of thrombin in the rat model of middle cerebral artery occlusion（ MCAO）,and to confirm the optimal observation time for pharmaceutical research. Methods： A total of 35 male SD rats were randomly divided into sham operation group and different time-phases groups after ischemia（ 1,6,12,24,48,and 72h）,with 5 rats in each group. A rat model of MACO was established using suture- occluded method in different time- phases groups after ischemia. Before the sacrifice of rats,neurologic impairment scores of rats were recorded in each group. The expression of thrombin mRNA in cerebral tissues of the rats was determined with fluorescence quantitative PCR assay,and the expression of thrombin protein in cerebral tissues of the rats was determined with Western blot. Results： The rats in the sham operation group showed no symptoms of neurologic impairment. The rats in the 1 to 72 h time- phases groups after ischemia showed adduction of contralateral forepaw,walking in circles,and falling to the contralateral side,most significant in the 24 h group. The expression of thrombin mRNA was increased 1h after cerebral ischemia and reached the peak value at24 h. The expression of thrombin protein was increased 1h after cerebral ischemia,and the increase continued to 24 h before it was stable at a high level. Conclusion： The expression levels of thrombin mRNA and protein in MCAO rats are increased after acute cerebral ischemia,and the peak values both appear at 24 h after ischemia,when the symptoms of neurologic impairment are also the most obvious. It indicates that thrombin may be involved in the pathological injury of acute cerebral ischemia,and 24 h after ischemic is the optimal observation time for pharmaceutical research.