【作者】 王晶；

【导师】 章军建；

【作者基本信息】 武汉大学 ， 临床医学， 2013， 博士

【摘要】 目的：通过构建慢病毒介导的HIF-1αshRNA载体,特异性地下调大鼠脑内HIF-1α的表达水平,观察HIF-1α在慢性脑低灌注状态下对认知功能的影响。方法：构建三种HIF-1αshRNA载体,使用慢病毒对其进行包装,测定病毒滴度。利用大鼠胶质瘤细胞筛选有效的干扰序列。随机将大鼠分为假手术+脑定位注射生理盐水组(sham组)、双侧颈总动脉结扎手术+脑定位注射生理盐水组(2VO组)、双侧颈总动脉结扎手术+脑定位注射慢病毒介导空载体组(2VO+RNAi negative组)、双侧颈总动脉结扎手术+脑定位注射慢病毒介导HIF-1αshRNA组(2VO+RNAi组)。采用物体识别试验、Morris水迷宫、荧光实时定量PCR、western-blot等方法分别检测大鼠的认知功能、HIF-1α基因及蛋白表达水平及认知相关分子CREB、 p-CREB、PSD95、MAP-2、SYN的蛋白表达水平。结果：与sham组大鼠相比,2VO组大鼠对新物体的识别能力下降,水迷宫实验逃避潜伏期延长。荧光实时定量PCR实验提示2VO+RNAi组较2VO+RNAi negative组海马区HIF-1α基因表达水平下调约40%；Western blot结果提示2VO术后的大鼠HIF-1α的表达增加,给予特异性RNAi干预后,2VO+RNAi组较2VO+RNAi negative组大鼠HIF-1α、CREB、p-CREB、MAP-2的蛋白表达水平下降,PSD95、突触素的蛋白表达水平无统计学差异。结论：慢性脑低灌注会引起大鼠认知功能损伤,应用RNAi技术对大鼠脑区特异性进行HIF-1α的下调,大鼠的认知功能进一步恶化。HIF-1α可能参与了慢性脑低灌注过程中对认知功能的调控过程。更多还原

【Abstract】 Objective To observe the impact of HIF-la on cognition in chronic cerebral hypoperfusion, we used the technology to synthetize lentivirus-mediated the HIF-lashRNA carrier decreased the level of expression of HIF-la in rat brain specifically.Methods We built three kinds of HIF-lashRNA, used lentivirus to package them. After measuring the virus titer, we used the rat glioma cells to identify the effective interference sequence. All the rats were randomly divided into sham-operated+injection of physiological saline (sham) group, bilateral carotid artery ligation+injection of physiological saline (2VO) group, bilateral carotid artery ligation+injection of empty vector (2VO+RNAi empty) group and bilateral carotid artery ligation+injection of HIF-lashRNA (2VO+RNAi) group. Object recognition test, Morris water maze, fluorescence real-time quantitative PCR, Western-blot and frozen sections were used to detect the cognition,the protein expression level of HIF-la, CREB, p-CREB, PSD95, MAP-2, SYN.Results Compared to sham rats, the ability of2VO rats to identify new objects was reduced. The escape latencies of2VO was extended. The fluorescence real-time quantitative PCR showed the level of HIF-la in2VO+RNAi group was lower than2VO+RNAi empty group. Western blot showed that the expression of HIF-1α in2VO group rats was increased. Compared to the2VO+RNAi empty group, the protein expression level of HIF-1α,CREB, p-CREB, MAP-2were reduced in2VO+RNAi group. The level of PSD95, SYN expression was almost same between the2VO+RNAi group and2VO+RNAi empty.Conclusion The chronic cerebral hypoperfusion in rats can cause cognitive impairment. By using RNAi to inhibit the HIF-la in the brain of rats, the cognition can be injured. HIF-la may be involved in the regulation of chronic cerebral hypoperfusion on cognition.更多还原