“中低温低流量”脑损伤SD大鼠模型的制作以及缺血预适应脑保护作用机制的研究

发布时间：2018-03-01 08:12

本文关键词： 中低温低流量动物模型脑缺血/再灌注损伤缺血预适应脑保护线粒体凋亡信号通路　出处：《南京医科大学》2014年博士论文　论文类型：学位论文

【摘要】：[目的] 制作成年雄性SD大鼠中低温低流量(MHLF)的脑损伤模型,进行表型分析,观察成年雄性SD大鼠模型中使用缺血预适应的策略进行脑保护的结果,并进一步研究其通过线粒体相关凋亡通路及其上游的信号通路发挥脑保护作用的机制。 [方法] 180只健康的成年雄性SD大鼠(8-10周龄,体重76-85g)随机的分为三组：假手术组、手术组和缺血预适应组,每组60只。手术组在SD大鼠的肛温降至(25±0.5)℃时阻断两侧颈总动脉120min后再重新开放,模拟临床上脑部中低温低流量的状态。假手术组除不阻断两侧颈总动脉外,其余的操作均相同。缺血预适应组在120分钟双侧颈动脉阻断前行缺血预适应,即阻断双侧劲总动脉2分钟后再灌注5分钟,反复循环4次,之后与手术组一样,阻断两侧颈动脉120分钟再重新开放。另取成年雄性健康SD大鼠(8-10周龄,体重76-85g)30只,随机的分为三组：假手术组、手术组和缺血预适应组,每组10只。利用激光多普勒血流仪监测假手术组、手术组和缺血预适应组(n=10)大鼠的局部脑血流量(rCBF),并记录在降温前、降温至(25.0±0.5)℃MHLF0～5min, MHLF10～15min、MHLF30～35min、MHLF55～60min、MHLF65～70min、 MHLF75～80min、MHLF85～90min、MHLF105～110min、MHLF115～120min和再灌注后0-5min,复温等十二个时间段的局部脑部血流量。对于前述180只雄性SD大鼠,我们根据再灌注后的时间随机的把每组60只雄性SD大鼠再分成5个亚组,每组12只：分别为再灌注后1h,6h,24h,72h和7d。通过TUNEL法检测三组SD大鼠脑组织的细胞凋亡水平,利用免疫组织化学与RT-PCR法检测各亚组对应的时间点中细胞色素C和caspase-3等线粒体凋亡通路相关蛋白的表达水平以及mRNA等表达变化情况,利用western blot检测细胞色素C、caspase-3与caspase-9以及其上游BCL-2与BAX以及AKT等蛋白的变化。 [结果] 成年雄性SD大鼠的局部脑血流量(rCBF)在手术组和缺血预适组的MHLF期间均下降了约84%左右。其手术组的rCBF、内环境等指标的变化趋势与临床MHLF中脑血流灌注水平,内环境变化趋势等基本一致。缺血预适应组的SD大鼠在再灌注6h,24h,72h和7d等各时间点死亡率均低于手术组,其脑组织病理改变较轻,脑组织损伤显著低于手术组,同时,TUNEL染色显示凋亡细胞数量在再灌注后6h,24h,72h和7d等各时间点较手术组均显著下降(P0.05or P0.01),并且免疫组化提示与手术组相比较,缺血预适应明显抑制了上述四个对应时间点的线粒体凋亡信号通路相关蛋白,细胞色素C与caspase-3的释放与活化等(P0.05or P0.01)。RT-PCR显示线粒体凋亡通路中的细胞色素C与caspase-3的mRNA表达水平在再灌注24h与72h表达水平较手术组明显减少(P0.05or P0.01)。 Western blot的结果显示在再灌注24h后细胞色素C与caspase-3的蛋白表达与上述结果一致,caspase-9在缺血预适应组的表达在再灌注24h后较手术组也显著减少(P0.05or P0.01)。在调控的上游基因中,缺血预适应组的BCL-2与AKT等蛋白的表达较之于手术组增强,而BAX的表达较手术组减弱(P0.05or P0.01)。 [结论] 本研究中制作的中低温低流量SD大鼠的脑损伤模型,其病理生理变化近似于临床上主动脉夹层手术中MHLF的脑部缺血再灌注的过程,模型制作成功。缺血预适应通过减少细胞DNA的断裂、阻止线粒体凋亡通路中细胞色素C的释放以及抑制了caspase-3、caspase-9等的活性从而发挥了脑保护的作用；同时,本实验研究发现其上游BCL-2、BAX以及AKT等基因与蛋白的相关变化,显示缺血预适应通过PI3K/Akt信号通路的这些节点抑制了线粒体凋亡通路的活性从而产生了脑保护作用的机制。缺血预适应可能成为临床上防治中低温低流量术后神经系统损伤新的治疗方法,而AKT蛋白可能成为新的分子治疗靶点。
[Abstract]:[Objective]
Production of adult male SD rats in low temperature and low flow rate (MHLF) of the brain injury model, phenotype analysis, observation of adult male SD rats were used in the model of ischemic preconditioning strategy of brain protection results, and further study the neuroprotective signaling through mitochondria related apoptosis pathway and its upstream mechanism.
[method]
180 healthy adult male SD rats (8-10 weeks old, weight 76-85g) were randomly divided into three groups: sham operation group, operation group and ischemic preconditioning group, 60 rats in each group. Group fall in rectal temperature of SD rats (25 + 0.5) on both sides of the common carotid artery occlusion after 120min C re open state simulation clinical brain hypothermia with low flow rate. The sham operation group was not blocked on both sides of the common carotid artery, the rest of the operation were the same. The preconditioning group blocked in 120 minutes before the bilateral carotid artery ischemic preconditioning, namely blocking bilateral common carotid arteries for 2 minutes after 5 minutes of reperfusion, repeatedly after 4 cycles, and the operation group, blocked on both sides of the carotid artery for 120 minutes and then re open. Another adult healthy male SD rats (8-10 weeks old, weighing 30 76-85g) were randomly divided into three groups: sham operation group, operation group and ischemic preconditioning group, 10 rats in each group. The use of laser Doppler flowmetry Monitoring of the sham operation group, operation group and ischemic preconditioning group (n=10) and local cerebral blood flow (rCBF), and recorded in the cool, cool to (25 + 0.5) MHLF0 C ~ 5min, MHLF10 ~ 15min, MHLF30 ~ 35min, MHLF55 ~ 60min, MHLF65 ~ 70min, MHLF75 ~ 80min. MHLF85 ~ 90min, MHLF105 ~ 110min, MHLF115 ~ 120min and 0-5min after reperfusion, local blood flow to the brain and rewarming time period twelve. For the 180 male SD rats, according to reperfusion time random each group of 60 male SD rats were divided into 5 subgroups in each group. 12: are 1h after reperfusion, 6h, 24h, 72h and 7d. by the level of apoptosis in brain tissue of rats with TUNEL assay in three SD group, the apoptosis pathway of cytochrome C and caspase-3 mitochondrial protein by immunohistochemistry and RT-PCR method corresponding to each subgroup of time points in the detection of expression level and mRNA Western blot was used to detect the changes in cytochrome C, caspase-3 and caspase-9, and the upstream BCL-2, BAX and AKT.
[results]
Regional cerebral blood flow in adult male SD rats (rCBF) during MHLF surgery group and ischemic preconditioning group were decreased by about 84%. The operation group of rCBF, the change trend of environmental indicators and clinical MHLF cerebral blood flow perfusion level, internal environment change trend of ischemic preconditioning is basically the same. SD rats at reperfusion 6H, 24h, 72h and 7d each time point of death were lower than the surgery group, the pathological changes of the brain is light, brain injury was significantly lower than the surgery group, at the same time, TUNEL staining showed that the number of apoptotic cells after reperfusion, 6h, 24h, 72h and 7d at each time point compared with the operation group were significantly decreased (P0.05or P0.01), and immunohistochemical staining showed that compared with the surgery group, ischemic preconditioning significantly inhibited the mitochondrial apoptosis pathway related proteins of the four at the same time, cytochrome C and caspase-3 release and activation of.R (P0.05or P0.01) T-PCR showed that the expression level of cytochrome C and caspase-3 mitochondrial apoptosis pathway in the expression of mRNA was significantly reduced compared with the operation group at 24h after reperfusion and 72h (P0.05or P0.01) Western blot. The results showed that in the reperfusion of cytochrome C and caspase-3 protein are consistent with the above results the expression of 24h, caspase-9 in ischemic preconditioning in after 24h compared with the operation group also significantly reduced the expression of group (P0.05or P0.01). In the upstream of gene regulation, ischemic preconditioning expression compared with operation group BCL-2 and AKT protein increased, while the expression of BAX decreased compared with the operation group (P0.05or P0.01).
[Conclusion]
Brain injury model in rats with low temperature and low flow SD in this study, the pathophysiological changes similar to clinical MHLF aortic dissection in brain ischemia reperfusion, ischemic preconditioning model was established successfully. The fracture reduction of cell DNA, prevent cytochrome C in the mitochondrial apoptosis pathway and inhibition of release the Caspase-3, caspase-9 activity and thus play the role of cerebral protection; at the same time, this study found that the upstream BCL-2, BAX and AKT related changes in gene and protein, showed that ischemic preconditioning mechanism through PI3K/Akt signal pathway of these nodes inhibits mitochondrial apoptotic pathway activity resulting in cerebral protective effect. Ischemic preconditioning may become clinically in prevention of hypothermia and low flow after injury to the nervous system a new treatment method, while AKT protein may become a new molecular therapeutic target.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R651.15;R-332

【共引文献】