本文選題：腦出血　切入點(diǎn)：NFATc4　出處：《南通大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的通過構(gòu)建腦出血(intracerebral hemorrhage,ICH)的在體和離體模型,研究活化T細(xì)胞核因子c4(nuclear factor of activated T-cells,cytoplasmic,calcineurin-dependent 4,NFATc4)在出血后的表達(dá)和分布變化,探討其對細(xì)胞凋亡的作用以及潛在的機(jī)制。方法通過大鼠斷尾,并借助腦立體定位儀,將抽取的自體全血注射到其右側(cè)腦部的尾狀核,以構(gòu)建腦出血在體模型。應(yīng)用行為學(xué)測試,包括前肢放置試驗(yàn)和轉(zhuǎn)角試驗(yàn),對造模后的大鼠進(jìn)行神經(jīng)功能評估和分析。在血腫周圍組織區(qū)域,NFATc4和相關(guān)分子,如Fas L等的蛋白表達(dá)均使用Western blot法檢測。運(yùn)用免疫組化方法進(jìn)一步探索NFATc4的表達(dá)和分布。通過免疫熒光雙標(biāo)法,發(fā)現(xiàn)NFATc4和神經(jīng)元之間存在共定位。此外,結(jié)合上述Western blot和免疫熒光這兩種方法,在離體和在體兩種情況下,繼續(xù)檢測Fas外源性凋亡途徑下游通路的表達(dá)和共定位情況,深入探討NFATc4與細(xì)胞凋亡之間的潛在關(guān)系和可能機(jī)制。兩個試驗(yàn)組的所有數(shù)據(jù)均使用Stata 7.0統(tǒng)計軟件進(jìn)行統(tǒng)計分析。結(jié)果在ICH的病理生理過程中,探索NFATc4的作用與機(jī)制。大鼠ICH模型構(gòu)建成功后,根據(jù)行為學(xué)測試評估發(fā)現(xiàn),腦出血大鼠表現(xiàn)出不同程度的神經(jīng)功能缺損。Western blot和免疫組化結(jié)果顯示,ICH后血腫周圍區(qū)域NFATc4的表達(dá)顯著上調(diào);免疫熒光結(jié)果提示NFATc4表達(dá)的變化主要體現(xiàn)在神經(jīng)元上,且存在亞細(xì)胞定位的變化,而核漿分離的結(jié)果證明NFATc4確實(shí)從細(xì)胞漿轉(zhuǎn)移到細(xì)胞核。NFATc4的表達(dá)增加伴隨著Fas配體(Fas L),活化的caspase-8和活化的caspase-3表達(dá)的上調(diào),并呈時間依賴性。此外,在神經(jīng)元中,NFATc4與活化的caspase-3存在共定位,表明NFATc4可能參與腦出血后的神經(jīng)元凋亡。為進(jìn)一步驗(yàn)證猜想,在血紅素刺激PC12細(xì)胞構(gòu)建的ICH離體模型中,驗(yàn)證了在體試驗(yàn)的結(jié)果,并采取干擾細(xì)胞中NFATc4表達(dá)的技術(shù),深入證實(shí)NFATc4可通過外源性途徑發(fā)揮其促進(jìn)細(xì)胞凋亡的作用。因此,ICH后NFATc4可以通過其促凋亡的作用加重大腦的繼發(fā)性損傷,這一觀點(diǎn)為后續(xù)的研究提供了作用靶點(diǎn)。結(jié)論總而言之,本研究首次檢測了腦出血后血腫周圍組織中NFATc4表達(dá)和分布的變化;所有的試驗(yàn)數(shù)據(jù)也都證實(shí),NFATc4參與腦出血后神經(jīng)元凋亡的過程。然而,仍需進(jìn)一步的研究以探索NFATc4促進(jìn)凋亡的潛在細(xì)胞和分子機(jī)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.
【學(xué)位授予單位】：南通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R743.34
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本文編號：1627505