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目的探讨凋亡抑制剂zVAD对脑缺血再灌注小鼠线粒体DNA拷贝数的影响。方法 80只雄性25~30gC57BL/6小鼠,随机分为大脑中动脉栓塞(MCAO)模型组(3组共24只)、模型给药组(3组共24只)、假手术组(3组共24只)和对照组(8只)。MCAO模型组应用线栓法,构建小鼠大脑中动脉栓塞模型并在1h后进行再灌注,模型给药组在再灌注前腹腔注射10mmol/L的ZVAD10mL/kg,假手术组暴露颈内动脉后缝合皮肤,对照组不进行任何操作。分别于再灌注后不同时间点(24、48、72h),对经手术操作的每组各8只小鼠进行改良神经功能损伤评分(mNSS),评分后取血约200μL并提取外周血基因组DNA,real-time PCR法检测相对线粒体DNA拷贝数。结果 MCAO模型组和模型给药组术后24、48和72h的mNSS评分均明显高于相应对照组(P<0.05),术后48、72h,模型给药组mNSS评分均明显低于相应MCAO模型组(P<0.05);术后24、48 h,MCAO模型组和模型给药组线粒体DNA拷贝数均高于对照组或假手术组,模型给药组高于MCAO模型组(P<0.05);术后72h,各组的线粒体DNA拷贝数差异无统计学意义(F=1.58,P>0.05)。结论凋亡抑制剂zVAD对脑缺血再灌注小鼠可起到一定的脑保护作用,这可能由于zVAD抑制了线粒体介导的凋亡通路,并通过端粒—线粒体途径促进了外周血线粒体DNA拷贝数的升高。
Objective To investigate the effect of zVAD, an inhibitor of apoptosis, on the copy number of mitochondrial DNA in cerebral ischemia-reperfusion mice. Methods Eighty male C57BL / 6 male mice (25-30 g) were randomly divided into MCAO model group (24 in 3 groups), model group (24 in 3 groups), 3 sham operation group A total of 24) and control group (8). The middle cerebral artery occlusion model was established in MCAO model group and reperfusion was performed after 1h. The model group was injected intraperitoneally with 10mmol / L ZVAD 10mL / kg before reperfusion and the carotid artery was exposed in sham operation group Suture the skin, the control group without any action. At each time point after reperfusion (24, 48, 72h), 8 rats in each group were operated to improve the neurological impairment score (mNSS). Blood samples were collected for about 200μL and the peripheral blood genomic DNA was extracted , real-time PCR method to detect relative mitochondrial DNA copy number. Results The mNSS scores of MCAO model group and model group at 24, 48, and 72 hours after operation were significantly higher than those of corresponding control group (P <0.05). At 48 and 72 hours after operation, the mNSS scores in model group were significantly lower than those in MCAO model group and model group (P <0.05). At 24 and 48 hours after operation, the copy number of mitochondrial DNA in MCAO model group and model group was higher than that in control group or sham operation group (P <0.05) ). There was no significant difference in copy number of mitochondrial DNA between groups at 72h (F = 1.58, P> 0.05). Conclusion The zVAD inhibitor may play a protective role in cerebral ischemia-reperfusion mice. This may be due to the fact that zVAD inhibits the mitochondria-mediated apoptotic pathway and promotes mitochondrial DNA in peripheral blood through telomere-mitochondrial pathway Increase in copy number.