在体大鼠心肌缺血再灌注中MMP-2/9、TIMP-1/2mRNA和蛋白表达及MMP-2/9酶活性的同步动态变化

Synchronal dynamic alterations of the expressions of MMP2/9、TIMP-1/2mRNA and protein and activities of MMP-2/9 in rat myocardium in vivo during ischemia-reperfusion

目的同步观察大鼠心肌缺血再灌过程中注基质金属蛋白酶(MMP-2)和MMP-9、基质金属蛋白酶抑制剂1(TIMP-1)和TIMP-2的mRNA和蛋白表达以及MMP-2和MMP-9的酶活性。方法80只大鼠随机分为8组:假手术组(Sham);单纯缺血30min组(I30min);再灌注1min组(R1min)、5min组(R5min)、10min组(R10min)、30min组(R30min)、再灌注1h组(R1h)和再灌注2h组(R2h),每组各10只。分别采用RT-PCR技术和免疫组化法检测心肌组织中MMP-2,-9和TIMP-1,-2的mRNA表达和蛋白表达;以酶谱分析法测血浆中MMP-2和MMP-9的酶活性。结果缺血再灌注过程中:(1)MMP-2、-9的mRNA表达呈升高趋势;MMP-2的mRNA表达在R1h组和R2h组分别与Sham组和I30min组比均显著差异;MMP-9的mRNA表达无明显变化;TIMP-1,-2的mRNA表达逐渐降低。MMP-2/TIMP-2,MMP-9/TIMP-1比值升高;(2)MMP-2,-9蛋白表达呈上升趋势,MMP-2蛋白表达于R2h达峰值;MMP-9蛋白表达R1h后开始显著升高;TIMP-1蛋白表达再灌注即刻即显著下降;TIMP-2蛋白表达于R5min开始显著下降;(3)MMP-2酶活性于R1min开始升高,R5min达峰值,R1h后逐渐降至假手术组水平;MMP-9酶活性呈升高趋势,R2h较Sham组呈显著差异。结论缺血再灌注过程中MMP-2,-9的mRNA与蛋白表达及酶活性较正常对照明显升高;TIMPs的mRNA和蛋白表达显著下调;MMPs/TIMPs比值失衡。MMPs/TIMPs比值失衡可能是治疗缺血再灌注损伤的一个潜在靶点。

Objective to observe synchronously alterations of the expression of MMP2/9、TIMP-1/2mRNA and protein and activities of MMP-2/9 in rat vivo myocardium during ischemia-reperfusion. Methods 80 rats were distributed randomly into 8 groups：sham ,simple ischemia for 30 minutes ,reperfusion for 1min,5min,10min,30min,1h and 2h. The expression of MMP-2,-9、TIMP-1,-2 mRNA and protein were determined by RT-PCR and immunohistochemistry,respectively. Activities of MMP-2,-9 in rats serum were detected by zymography. Results （1） The expression of MMP-2,-9 mRNA tended to increase during ischemia reperfusion. There was significant difference of MMP-2 mRNA expression in Rlh and R2 t, compared wltb Sham and 130rain, respectively, but there was no difference of MMP-9 mRNA expression in the whole I/R process. The expression of TIMP-1 ,-2 mRNA were presented to decrease. The ratio of MMP- 2/TIMP-2 and MMP-9/TIMP-1 were increased. （2） The expression of MMP-2, -9 protein had a trend of increase ; The expression of MMP-2 protein was peaked at 2h of reperfuslon. From 1h of reperfusion, there was a marked increase in expression of MMP-9 protein. From the beginning of repeffusion, the expression of TIMP-1 protein was presented significant decrease, and that of TIMP-2 protein was decreased obviously at the beginning of R5min. （3）The activity of MMP-2 began to increase at lmin of reperfusion, peaked at 5rain and gradually decreased to tbe level of group sham after 1tl of reperfusion. The activity of MMP-9 tended to increase. There was marked difference in R2h, compared with sham. Conclusions During myocardial ischemia-reperfusion, the mRNA and protein expressions and the activities of MMPs were increased signifi- cantly, the mRNA and protein expressions of TIMPs were downregulated dramatically, the balance of MMPs/ TIMPs was impaired. It was concluded that disbalance of MMPs/TIMPs may therefore be a potential target of treatment of myocardial reperfusion injury.