ApoE基因敲除构建大鼠缺血性脑卒中模型的实验研究

Experimental Study of ApoE Gene Knockout in Rats with Ischemic Stroke Model

目的探讨采用载脂蛋白E(ApoE)基因敲除构建稳定的大鼠缺血性脑卒中的过程。方法选用健康的6周龄雄性ApoE基因敲除大鼠100只,SPF级SD雄性大鼠100只,依据大鼠种类分为两组。用线栓法堵塞大脑中动脉2h,缺血后2h向外拔出线栓约2mm再灌注22h后行Zea-Longa评分,评分在2-3分为成功模型。统计不同种类大鼠成模成功率情况;在模型构建3d、7d时行神经功能缺损严重程度评分(NSS)和横木行走实验(BWT)评分评估模型的稳定性,同时行核磁共振(MRI)检查了解各处理梗塞病灶范围及变化;再通后灌注7d断头取脑,进行氯化三苯基四氮唑(TTC)染色及荧光染色观察梗塞范围变化情况。结果ApoE基因敲除大鼠与SD大鼠两组模型3d内成功率高,分别为76%和77%,7d内模型成功率分别为75%和73%,两组无统计学差异(P>0.05)。ApoE基因敲除大鼠大脑中动脉阻塞(MCAO)模型缺血再通灌注7d时NSS和BWT评分较3d时稍减低,但无统计学差异;SD大鼠模型组在缺血再通灌注7d时NSS和BWT评分较3d时稍减低,但有显著统计学差异(P<0.001)。MR影像显示缺血区域呈现明显高信号,随时间推移两组动物模型缺血范围呈先扩大后缩小趋势,SD组大鼠模型缺血范围较ApoE基因敲除组大。TTC染色缺血区染染成苍白色。ApoE基因敲除模型组荧光标记显示神经细胞大小、分布较SD组均匀,阳性细胞数明显高于SD组(P<0.05)。结论ApoE基因敲除大鼠可用于制备稳定的MCAO模型。

Objective To investigate the process of apolipoprotein E(ApoE)gene knockout to construct stable ischemic stroke in rats.Methods 6 weeks old ApoE gene-knockout rats(n=100)and Sprague-Dawley rats(n=100)were selected to establish an animal model of ischemic stroke.These rats were divided into two groups according to the rat species.The middle cerebral artery was blocked with suture method to prepare cerebral ischemia reperfusion model.After cerebral ischemia for two hours and reperfusion for 22 nd hours,the neurological function score of rats was scored according to the Zea-Longa scale.The Zea Longa 5 scoring scale,NSS and BWT were used to estimate the neurological deficiency while TTC staining method and MRI were used to measure and calculate the volume of cerebral infarction for all of the animal models.The percentage of successful models with 2-3 grade scorings and the coefficient of the variations of cerebral infarct volume were used to estimate the stability of the models.Results The rate of success of establishment models and the percentage of establishment models with 2-3 grade neurological scores in experimental group were not higher compared with control group(P>0.05).The success rate of ApoE gene-knockout rats at 3 and 7 days was 76%and 75%,and SD rats at 3 and 7 days was 76%and 75%.The NSS and BWT of ApoE gene-knockout rats model at 7 days were slightly lower compared with it at 3 days(P>0.05)and SD rats model at 7 days were significantly lower compared with it at 3 days(P<0.05).The MR images showed that the signal of the ischemic region was significantly higher than contralateral symmetry area.The ischemic range of two groups were slightly increased at the first3 days after cerebral ischemia reperfusion,and achieved the peak at the third day,which decreasing then.The ischemic range of the SD group was larger than that of the ApoE knockout group.The?area?of the infarcted tissues which were stained white or pale by TTC.The numbered of nerve cells in experimental group were significantly higher compared with control group(P<0.05).Conclusion ApoE gene-knockout rats can be used to prepare for establishing stable MCAO models.