CAP1在创伤性颅脑损伤后的表达及其在星形胶质细胞增殖中的功能研究

发布时间：2018-05-13 08:04

本文选题：颅脑损伤 + CAP1　；参考：《南通大学》2015年硕士论文

【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。
[Abstract]:Objective to study the expression and distribution of cyclase associated protein (1(Cyclase-associated protein 1) and its potential mechanism after brain injury in rat brain injury model and primary astrocyte proliferation model in vitro. So as to find a new way for the clinical treatment of traumatic brain injury. Methods 1. The cerebral cortex mRNA and protein were extracted from the normal group and the injured group. The expression of CAP1 was detected by RT-PCR Western Blot and immunohistochemistry, and the expression of CAP1 in different cells and colocalization with Ki-67 was detected by double immunofluorescence method. The relationship between the expression of CAP1 and brain injury was preliminarily determined. The astrocytes were cultured in vitro, and the inflammatory activation model of astrocytes induced by LPS was established. Western Blot was used to detect the expression of CAP1 and other related proteins at different time points of stimulation. The correlation between CAP1 and astrocyte proliferation was determined. The expression of CAP1 in astrocytes was interfered by specific molecular interference RNAs, and the inflammatory activation model was constructed. The effect of CAP1 expression on astrocyte proliferation after craniocerebral injury was further determined by cell proliferation test and cell flow analysis. Results 1. The results of RT-PCR and Western Blot showed that the expression of CAP1 in cerebral cortex began to up-regulate 12 hours after traumatic brain injury, and gradually increased to the peak at 7 days. The expression of CAP1 was increased in injured tissues. The results of immunofluorescence double labeling analysis showed that there was a co-localization of CAP1 with astrocytes, and the expression of CAP1 was up-regulated in astrocytes after injury. At the same time, the expression of CAP1 was consistent with that of Ki-67, and there was colocalization of .2in astrocytes. In vitro, LPS was used to stimulate primary astrocytes, and the expression of PCNA and cyclin were detected at different time points. It was found that the expression of CAP1 increased gradually with the prolongation of the action time, and the expression of CAP1 increased gradually. In the model of LPS induced by intervention of CAP1 expression, the cell growth was inhibited and cell cycle was blocked in the interference group. Conclusion the expression of CAP1 is up-regulated in astrocytes after traumatic brain injury. In the process of brain injury, CAP1 may be involved in the process of brain injury by regulating the activation of cell cycle in astrocytes and inducing the proliferation of astrocytes.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R651.15

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