【作者】 张海燕；

【导师】 程纯； 沈爱国；

【作者基本信息】 南通大学 ， 免疫学（神经免疫学）， 2015， 硕士

【摘要】 目的通过构建大鼠颅脑损伤模型以及原代星形胶质细胞体外增殖模型,研究环化酶相关蛋白1(Cyclase-associated protein 1,CAP1)在颅脑损伤后的表达和分布变化,探讨其对细胞增殖的作用以及潜在的机制,从而为创伤性颅脑损伤的临床治疗寻找新途径。方法1、建立大鼠颅脑损伤模型,提取正常组及损伤组大鼠大脑皮层mRNA和蛋白。运用RT-PCR、Western Blot和免疫组化方法检测CAP1的表达变化;运用双重免疫荧光方法检测颅脑损伤后CAP1在不同细胞以及与Ki-67的共定位情况。初步明确CAP1的表达变化与颅脑损伤的相关性。2、体外培养大鼠星形胶质细胞,构建LPS诱导的星形胶质细胞炎性活化模型,用Western Blot检测CAP1、PCNA等相关蛋白在刺激的不同时间点的表达变化,明确CAP1与星形胶质细胞增殖的相关性。3、利用特异性分子小干扰RNA,干扰星形胶质细胞CAP1的表达,并构建炎性活化模型,通过细胞增殖能力检测和细胞流式分析,进一步明确CAP1的表达对颅脑损伤后星形胶质细胞增殖的影响。结果1、大鼠创伤性颅脑损伤模型中,RT-PCR与Western Blot结果显示损伤12h后大脑皮层中CAP1的表达开始上调,并逐渐升高,7d达到高峰;免疫组化检测结果与之相一致,CAP1在损伤组织中表达量增加。免疫荧光双标记分析结果显示:CAP1与星形胶质细胞细胞存在共定位,且损伤后CAP1在星形胶质细胞中表达上调。同时,CAP1与Ki-67表达存在时间上的一致性,且在星形胶质细胞存在共定位。2、体外研究中,运用LPS刺激原代星形胶质细胞,在不同的时间点检测PCNA及细胞周期相关蛋白的表达变化,发现随着作用时间的延长,其表达量也逐渐升高,在此过程中,CAP1的表达量也逐渐升高。3、在干预CAP1的表达后的LPS诱导模型中,干扰组细胞生长受到抑制,细胞周期出现阻滞。结论创伤性脑损伤后CAP1的表达上调,主要表达在星形胶质细胞中。在颅脑损伤过程中,CAP1可能通过调节星形胶质细胞中的细胞周期的激活,诱导星形胶质细胞的增殖,从而参与颅脑损伤的过程。更多还原

【Abstract】 Objective To investigate the expression and localization changes of Cyclaseassociated protein 1(CAP1) in the brain cortex after traumatic brain injury and clarify the role of CAP1 in the processes of central nervous system(CNS) injury.Methods Sprague-Dawley(SD) rats were subjected to traumatic brain injury. Expression patterns of CAP1 were observed in the injured cortex by RT-PCR, Western Blot, immunohistochemistry and the distribution of CAP1 was detected by double immunofluorescence labeling assay.In vitro, primary rat astrocytes were prepared. Then, inflammatory activation model was constructed by LPS. The expression patterns of CAP1, PCNA and other related proteins were detected at different time point by Western Blot to clear the correlation between CAP1 with astrocyte proliferation.By using CAP1 specific siRNA to knock down the expression of CAP1 of astrocytes in the inflammatory activation model, we further clarify the effect of CAP1 on the proliferation of astrocytes after brain injury through the cell proliferation and cell cycle test.Results1. In the rat traumatic brain injury model, Western Blot results showed that the expression of CAP1 in cerebral cortex began to increase at 12 h, and reached the peak at 7d; immunohistochemistry results also showed an identical increase of CAP1 in injured group. Double immunofluorescence labeling analysis showed that CAP1 was mainly increased in astrocytes. At the same time, CAP1 was co-localization with Ki-67 in astrocytes.2. In vitro, after treating primary astrocytes with LPS at different time points, we found the expression of PCNA and cell cycle related proteins were gradually increased. During this process, the expression of CAP1 was also gradually increased.3. Silencing CAP1 by specific siRNA inhibited astrocytes growth and cell cycle partly.Conclusions After traumatic brain injury, the expression of CAP1 is upregulated mainly in astrocytes. CAP1 may be involved in the activation and proliferation of astrocytes during the process of traumatic brain injury.更多还原