NFATc4参与脑出血后的神经元凋亡

发布时间：2018-03-18 02:08

  本文选题：脑出血　切入点：NFATc4　出处：《南通大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的通过构建脑出血(intracerebral hemorrhage,ICH)的在体和离体模型,研究活化T细胞核因子c4(nuclear factor of activated T-cells,cytoplasmic,calcineurin-dependent 4,NFATc4)在出血后的表达和分布变化,探讨其对细胞凋亡的作用以及潜在的机制。方法通过大鼠断尾,并借助脑立体定位仪,将抽取的自体全血注射到其右侧脑部的尾状核,以构建脑出血在体模型。应用行为学测试,包括前肢放置试验和转角试验,对造模后的大鼠进行神经功能评估和分析。在血肿周围组织区域,NFATc4和相关分子,如Fas L等的蛋白表达均使用Western blot法检测。运用免疫组化方法进一步探索NFATc4的表达和分布。通过免疫荧光双标法,发现NFATc4和神经元之间存在共定位。此外,结合上述Western blot和免疫荧光这两种方法,在离体和在体两种情况下,继续检测Fas外源性凋亡途径下游通路的表达和共定位情况,深入探讨NFATc4与细胞凋亡之间的潜在关系和可能机制。两个试验组的所有数据均使用Stata 7.0统计软件进行统计分析。结果在ICH的病理生理过程中,探索NFATc4的作用与机制。大鼠ICH模型构建成功后,根据行为学测试评估发现,脑出血大鼠表现出不同程度的神经功能缺损。Western blot和免疫组化结果显示,ICH后血肿周围区域NFATc4的表达显著上调;免疫荧光结果提示NFATc4表达的变化主要体现在神经元上,且存在亚细胞定位的变化,而核浆分离的结果证明NFATc4确实从细胞浆转移到细胞核。NFATc4的表达增加伴随着Fas配体(Fas L),活化的caspase-8和活化的caspase-3表达的上调,并呈时间依赖性。此外,在神经元中,NFATc4与活化的caspase-3存在共定位,表明NFATc4可能参与脑出血后的神经元凋亡。为进一步验证猜想,在血红素刺激PC12细胞构建的ICH离体模型中,验证了在体试验的结果,并采取干扰细胞中NFATc4表达的技术,深入证实NFATc4可通过外源性途径发挥其促进细胞凋亡的作用。因此,ICH后NFATc4可以通过其促凋亡的作用加重大脑的继发性损伤,这一观点为后续的研究提供了作用靶点。结论总而言之,本研究首次检测了脑出血后血肿周围组织中NFATc4表达和分布的变化;所有的试验数据也都证实,NFATc4参与脑出血后神经元凋亡的过程。然而,仍需进一步的研究以探索NFATc4促进凋亡的潜在细胞和分子机制。
[Abstract]:Objective to study the expression and distribution of activated T nuclear factor of activated T-cell factor after intracerebral hemorrhage (ICH) in vivo and in vitro, and to explore its effect on apoptosis and its underlying mechanism. The whole blood was injected into the caudate nucleus on the right side of the brain with the help of the brain stereotactic locator to construct the model of intracerebral hemorrhage in vivo. The behavior test, including the forelimb placement test and the rotation test, was used to establish the model of intracerebral hemorrhage in vivo. The neural function of rats was evaluated and analyzed. NFATc4 and related molecules were found in the tissue around hematoma. For example, the protein expression of Fas L was detected by Western blot method. The expression and distribution of NFATc4 were further explored by immunohistochemical method. The co-localization between NFATc4 and neurons was found by immunofluorescence double labeling method. Combined with the above two methods, Western blot and immunofluorescence, the expression and co-localization of downstream pathway of exogenous apoptotic pathway of Fas were detected in vitro and in vivo. The potential relationship and possible mechanism between NFATc4 and apoptosis were studied in depth. All the data of the two groups were statistically analyzed by Stata 7.0 statistical software. Results in the pathophysiological process of ICH, To explore the role and mechanism of NFATc4. After the successful construction of rat ICH model, we found that, The results of Western blot and immunohistochemistry showed that the expression of NFATc4 in the perihematoma region after ICH was significantly up-regulated, and the expression of NFATc4 was mainly reflected in the neurons of rats with intracerebral hemorrhage. The results of nuclear and cytoplasmic segregation showed that the expression of NFATc4 from cytoplasm to nucleus increased with the up-regulation of Fas ligand FAS L, activated caspase-8 and activated caspase-3 in a time dependent manner. The co-localization of NFATc4 and activated caspase-3 in neurons suggests that NFATc4 may be involved in neuronal apoptosis after intracerebral hemorrhage. In order to further verify the conjecture, the results of in vivo test were verified in the ICH model constructed by heme stimulated PC12 cells. By interfering with the expression of NFATc4 in cells, it is proved that NFATc4 can promote apoptosis through exogenous pathway. Therefore, NFATc4 can increase the secondary injury of brain by its effect of promoting apoptosis. Conclusion in conclusion, the expression and distribution of NFATc4 in perihematoma tissues after intracerebral hemorrhage were detected for the first time. All the experimental data have confirmed that NFATc4 is involved in neuronal apoptosis after intracerebral hemorrhage. However, further research is needed to explore the potential cellular and molecular mechanisms of NFATc4 promoting apoptosis.
【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.34
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本文编号：1627505