【作者】 万群；

【导师】 万琪；

【作者基本信息】 第四军医大学 ， 神经病学， 2004， 硕士

【摘要】 脑缺血再灌注早期，损伤区域会迅速产生大量的氧自由基，通过直接或间接的作用引起神经元DNA氧化损伤，包括DNA碱基损伤、单链断裂和双链断裂。这种氧化性DNA损伤可以通过自身DNA修复系统进行修复，但是，如果DNA损伤持久或严重，超出自身修复能力，则诱导神经元死亡。研究表明脑缺血再灌注早期DNA修复酶的表达减少可能与随后发生的细胞死亡有关。神经细胞的死亡是脑缺血后神经功能受损的基础，对DNA损伤修复的研究有可能深入揭示缺血神经元的死亡机制，为缺血性脑血管病的治疗提供新的策略。缺血预处理和中药丹参对于缺血性脑损伤都有显著的神经保护作用，关于二者的脑保护机制已有多方面的探索，但仍未完全明确，是否可能通过调节内源性DNA修复活性而发挥保护作用还有待进一步研究。 本研究采用大脑中动脉线拴法建立大鼠局灶性脑缺血再灌注模型，利用HE染色方法、免疫组织化学方法和原位末端标记法(TUNEL法)，首先观察大脑中动脉阻塞(MCAO)2h再灌注0.5h、2h、6h、12h、24h、48h后缺血区额顶叶皮质和尾壳核DNA修复蛋白Ku70的表达及神经元凋亡情况，分析二者的变第四军医大学硕士学位论文化在时间上的关系;然后分别观察缺血预处理和中药丹参对脑缺血再灌注后Ku7O表达的影响，初步探讨脑缺血再灌注早期DNA修复酶Ku7O的表达变化规律及其意义，并从DNA损伤修复的角度探讨缺血预处理和中药丹参对抗脑缺血再灌注损伤的分子机制。 实验结果如下: (l)大鼠大脑中动脉阻塞再灌注(MCAO服)后损伤区域主要位于额叶皮质、顶叶皮质和尾壳核。 (2)正常对照组、假手术组及缺血再灌注对侧额顶叶皮质、尾壳核可见Ku70广泛表达，定位于细胞核:MCAOZh、再灌注0.5h，缺血侧尾壳核Ku7O阳性细胞数开始减少(P<0.05)，再灌注Zh，额顶叶皮质Ku70阳性细胞数开始减少(尸<0.05)，于再灌注6h，上述区域Ku70表达水平显著降低(P<0.01)，并持续至再灌注48h;实施缺血预处理或给予腹腔注射丹参后，明显抑制了Ku7O表达水平的降低，各时间点Ku7O阳性细胞数均较相应对照组明显升高(尸<0.01)。 (3)正常对照组、假手术组及缺血再灌注对侧未见TUNEL阳性细胞:‘MCAOZh、再灌注0.sh一2h，缺血区偶见散在的TUNEL阳性细胞:再灌注6h，TUNEL阳性细胞数开始增多(P<0.01)，随着再灌注时间的延长，阳性细胞数不断增高，再灌注24h和48h，TUNEL阳性细胞数达到高峰(尸<0.01):实施缺血预处理或给予丹参腹腔注射，明显抑制了脑缺血再灌注后细胞凋亡的发生，各时间点TUNEL阳性细胞数均较相应对照组显著减少 (P<0 .01)。(4) Ku蛋白在损伤区域内全面减少发生在缺血性神经元凋亡高峰出现之前，二者呈负相关关系。第四军医大学硕士学位论文综上，本研究的初步结论如下: (1)脑缺血再灌注早期，损伤区域Ku蛋白表达下降，可能是导致DNA双链断裂无法得到修复、细胞随之死亡的机制之 (2)缺血预处理可能通过上调DNA修复蛋白Ku的表达或括性而增强DNA修复能力，从而发挥其内源性神经保护作用。 (3)丹参可能通过抑制缺血再灌注后DNA修复蛋白Ku的减少，保护机体自身DNA修复功能免遭破坏而发挥其神经保护作用。更多还原

【Abstract】 A large number of free radicals are produced in a burst-like manner at the early stage of cerebral ischemia/reperfusion and mainly cause oxidative DNA damage. Neuronal DNA damage caused by oxidative stress consistes of the lesion of base, DNA single strand breaks (SSB) and DNA double strand breaks (DSB). Oxidative DNA damage after ischemia / reperfusion can be repaired by DNA repair system, but continuous or severe DNA damage against DNA repair could induce neuronal death, such as necrosis or apoptosis. It has been reported that early decrease of DNA repair enzyme probably has correlation with subsequent DNA-damaged cell death after cerebral ischemia / reperfusion. As neuronal death was the basis of brain injury, researches on DNA damage and repair would probably reveal the mechanism of ischemic neuronal death, and will bring a new strategy for treating ischemic cerebrovascular diseases. It has been proved that both ischemic preconditioning (IPC) and radix salviae miltiorrhizae (RSM) have significantneuroprotection against ischemic injury in the brain. A number of studies have been performed to clarify the protective effect and mechanism, however, whether IPC and RSM have any effects on the DNA damage and repair in the neuropathological process following cerebral ischemia/reperfusion still remains to be investigated.In this study, we made the model of rat middle cerebral artery occlusion/ reperfusion and used H&E stain method, immuno-histochemical technology and TUNEL detection, in order to explore the relationship between the expression of Ku70 protein-which is critical to the repair of DNA DSB and neuronal death after focal cerebral ischemia/reperfusion, and the possible molecular mechanisms by which IPC and RSM protect brain. Firstly, we observed the expression of Ku70 protein and neuronal apoptosis in the ischemic cortex and caudatum after MCAO 2h reperfusion 0.5h, 2h, 6h, 12h, 24h, 48h, and evaluated the temporal relationship between Ku70 protein and neuronal apoptosis. Then we investigated the influence of IPC and RSM on the expression of Ku70 protein after focal cerebral ischemia/reperfusion.Our results showed:(1) HE stain indicated the model of rat MCAO/R was credible, and the damaged regions included cortex and caudate putamen.(2) Immunohistochemical method showed the nuclear expression of Ku70 protein in the nomal control group, sham operation group and controlateral nonischemic hemisphere. The expression of Ku70 started to decrease at 0.5h of reperfusion in the caudate putamen and at 2h in the cortex (P<0.05), The number of Ku70 positive cells was decreased remarkably in the entire damaged area as early as 6hours after reperfusion and remained reduced until 48 hours. IPC (MCAO 20min) or RSM (15g/kg i.p.) significantly suppressed the early reduction of Ku70 expression in ischemic cortex and caudate putamen, compared with controls, the number of Ku70 positive cells was significantly higher at different reperfusion time (P<0.01).(3) There were no TUNEL positive cells in the nomal control group, sham operation group and controlateral nonischemic hemisphere. TUNEL positive cells begin to rise from 6h after reperfusion, reached peak at 24h and 48h (P<0.01). IPC (MCAO 20min) or RSM (15g/kg i.p.) reduced significantly TUNEL positive cells in ischemic cortex and caudate putamen at different reperfusion time after MCAO 2h (P<0.01).(4) The number of Ku 70 positive cells was decreased remarkably in the entire damaged area as early as 6 hours after ischemia/reperfusion and neuronal apoptosis occurred after the reduction of Ku expression.Our results demonstrate:(1) Expression of DNA repair protein Ku70 decreased remarkably in the damaged regions at early stage of cerebral ischemia/reperfusion, which results in DNA DSB unrepaired or incompletely repaired, It may involve the mechanism of subsequent neuronal apoptosis.(2) The neuroprotective mechanism of IPC is associated with upregulating expression or activity of DNA repair更多还原