發(fā)布時間：2018-05-08 02:11

  本文選題：Nrf2 + 自噬�。� 參考：《山東大學(xué)》2016年博士論文

【摘要】：研究背景：轉(zhuǎn)錄因子NE-2相關(guān)因子2(Nrf2)是堿性亮氨酸拉鏈轉(zhuǎn)錄因子Cap'n'Coall(CNC)家族的成員,Nrf2通過與一種順式增強(qiáng)子序列結(jié)合,我們稱之為抗氧化反應(yīng)元件(ARE),其核心核苷酸序列為'5-RTGACNNNGC-3',與這種增強(qiáng)子結(jié)合可啟動200多種歸屬于不同領(lǐng)域的基因的表達(dá),包括抗氧化基因、Ⅱ相解毒酶、轉(zhuǎn)錄因子、轉(zhuǎn)運(yùn)蛋白、清道夫受體以及分子伴侶蛋白等等。因此,Nrf2具有廣泛而復(fù)雜的生物學(xué)功能,從經(jīng)典的抗氧化防御,到細(xì)胞周期的調(diào)控和蛋白質(zhì)量的控制。Nrf2對心臟的保護(hù)作用已經(jīng)在許多動物模型中證實,包括以主動脈弓縮窄術(shù)(TAC)引發(fā)心臟壓力負(fù)荷增加誘導(dǎo)的失代償性心臟重構(gòu)和心功能障礙模型。從分子水平上分析,轉(zhuǎn)錄因子Nrf2啟動抗氧化蛋白以及解毒蛋白的表達(dá),從而對抗氧化應(yīng)激壓力導(dǎo)致的心臟損傷和功能失常。同時,研究發(fā)現(xiàn)Nrf2可促進(jìn)自噬清除毒性的多聚泛素化蛋白聚集體的功能,從而保護(hù)心臟不受細(xì)胞內(nèi)毒性蛋白的損傷。但是,與之前的結(jié)論相反,最近的研究發(fā)現(xiàn),在一種蛋白聚集誘發(fā)心肌病的小鼠動物模型中,這種模型通過向小鼠基因組內(nèi)導(dǎo)入人來源的alpha B-晶狀體蛋白基因突變體(hCryABR120G)并進(jìn)行小鼠老齡化實現(xiàn),敲除Nrf2基因可以抵御心臟的還原壓力,減少多聚泛素化蛋白在心臟內(nèi)的聚集,減緩心臟病理性肥大,改善心力衰竭。分析原因,持續(xù)性的Nrf2激活可能引發(fā)過度的還原壓力導(dǎo)致hCryABR120G誘發(fā)的心肌病。然而,也有研究發(fā)現(xiàn)心肌特異性敲除Nrf2基因的小鼠對抗了TAC術(shù)后4周的壓力負(fù)荷誘發(fā)的失代償性心臟重構(gòu)和功能障礙,這與Nrf2介導(dǎo)還原壓力的理論相違背。另一方面,前期的研究也證明在自噬功能障礙的狀態(tài)下,Nrf2的激活導(dǎo)致了肝臟的損傷。另外,自噬功能障礙在小鼠CryABR120G (mCryABR120G)基因誘導(dǎo)的心肌病中起了重要作用,mCryABR120G基因和hCryABR120G基因有著幾乎相同的核苷酸序列,且衰老本身也伴隨著自噬功能不足或損傷,從這些關(guān)聯(lián)上看,衰老的hCryABR120G小鼠心臟中Nrf2介導(dǎo)的負(fù)面作用很可能與自噬功能不足有關(guān)。綜上所述,在不同的病理狀態(tài)下,Nrf2對心臟的作用可能是保護(hù)性的也可能是損傷性的,這種雙面作用確切的機(jī)制目前還沒有研究清楚。在本次研究中,我們發(fā)現(xiàn)心臟中Nrf2基因激活的病理生理學(xué)作用與自噬功能的完整性密切相關(guān)。自噬功能完整時Nrf2的激活對心臟的適應(yīng)性反應(yīng)起著重要作用,然而,在自噬功能障礙時,Nrf2的激活又介導(dǎo)了心臟的失代償性重構(gòu)和功能障礙。我們在研究中發(fā)現(xiàn),自噬功能損傷可能是通過阻斷核內(nèi)Fyn控制的Nrf2向細(xì)胞核外的轉(zhuǎn)運(yùn),從而導(dǎo)致了Nrf2在核內(nèi)積聚,啟動血管緊張素原的大量表達(dá),進(jìn)而惡化心臟失代償性重構(gòu),加重心功能損傷。研究目的：1、研究Nrf2在TAC手術(shù)誘發(fā)的壓力負(fù)荷引起的心臟重構(gòu)和心功能障礙的病理進(jìn)展過程中所起的作用；2、研究Nrf2激活對心臟的作用與自噬功能狀態(tài)之間的關(guān)系；3、探討在不同自噬功能狀態(tài)下,Nrf2影響心臟功能的具體機(jī)制。研究方法：第一部分：1、用Nrf2基因敲除小鼠和野生型對照組小鼠進(jìn)行主動脈弓縮窄手術(shù),建立心衰模型,檢測術(shù)后存活狀況,在模型的早期EBD檢測心肌壞死,模型的晚期階段,超聲、qPCR、WGA、Masson、免疫組化檢測心臟重構(gòu)狀況和心功能,分析不同病理生理學(xué)階段Nrf2基因敲除對心臟的影響；2、通過對TAC術(shù)后的WT小鼠術(shù)后不同時間點進(jìn)行自噬流的檢測,分析TAC術(shù)后不同階段自噬功能的狀態(tài);3、運(yùn)用心肌特異性Atg5基因敲除小鼠建立自噬功能障礙心臟的TAC模型,觀察在自噬功能障礙時心臟的病理狀態(tài)以及Nrf2和其下游基因表達(dá)水平。第二部分：1、以qPCR和Western Blot的方法檢測白噬功能障礙小鼠心臟承受壓力負(fù)荷時心衰相關(guān)基因Agt的表達(dá)狀況；2、以Western Blot的方法檢測自噬功能障礙對小鼠心臟承受壓力負(fù)荷時各信號通路激活水平的影響,并與Nrf2基因敲除小鼠心臟壓力負(fù)荷模型相對比,分析兩者之間的聯(lián)系；3、建立Nrf2/Atg5雙敲小鼠心臟壓力負(fù)荷動物模型,以免疫組化、western blot檢測更具體的分析Nrf2對壓力誘導(dǎo)心衰的病理生理作用與自噬功能的關(guān)系。研究結(jié)果：第一部分：1、Nrf2基因敲除加重了TAC手術(shù)誘發(fā)的心臟急性損傷,但使手術(shù)引起的遠(yuǎn)期的心臟重塑和心力衰竭得到了緩解；2、在壓力負(fù)荷引起的心臟病程中,Nrf2激活所起的病理生理學(xué)作用與自噬功能的完整性有關(guān)；3、自噬功能障礙的小鼠心臟壓力負(fù)荷增加時,存在Nrf2的過度表達(dá)和激活。第二部分：1、在自噬功能缺失的壓力超負(fù)荷心臟中,Nrf2啟動Agt的表達(dá)；2、在壓力負(fù)荷誘導(dǎo)損傷的心臟,自噬功能障礙阻斷了TAC誘導(dǎo)的Jak/Fyn通路的激活,使Nrf2向細(xì)胞核外轉(zhuǎn)運(yùn)降解減少。研究結(jié)論：1、自噬功能完整時Nrf2激活對心臟起保護(hù)作用,而當(dāng)自噬功能障礙時,Nrf2的激活加速了心臟病理性重構(gòu)和心功能失常的進(jìn)程；2、在自噬功能完整時,Nrf2激活可以降低心肌壞死率保護(hù)心臟,但在自噬功能不足時,Nrf2激活了心臟內(nèi)血管緊張素Ⅱ通路,使病情進(jìn)一步惡化；3、自噬功能的障礙導(dǎo)致了Nrf2在細(xì)胞核內(nèi)的聚集,進(jìn)一步啟動了血管緊張素原的高表達(dá),Nrf2在核內(nèi)的聚集與Jak2/Fyn信號通路的阻斷有關(guān)。
[Abstract]:Background : The transcription factor NE - 2 - associated factor 2 ( Nrf2 ) is a member of the basic leucine zipper transcription factor Cap ' n ' Coall ( CNC ) family . Nrf2 binds to a sequence of cis - enhancer sequences , and we call it anti - oxidation reaction element ( ARE ) whose core nucleotide sequence is ' 5 - RTMM NNlngc - 3 . It is found that Nrf2 plays an important role in preventing cardiac injury and dysfunction induced by stress stress in mice . The purpose of this study was to study the role of Nrf2 in the pathogenesis of cardiac remodeling and cardiac dysfunction induced by TAC operation .
2 . To study the relationship between the activation of Nrf2 and the function of autophagy in the heart ;
3 . To investigate the specific mechanism of Nrf2 on cardiac function in different autophagy states . The study was as follows : 1 . Using Nrf2 gene knockout mice and wild - type control group mice to perform aortic arch narrowing operation , establish a heart failure model , detect the survival status of the model , detect myocardial necrosis at the early stage of the model , detect cardiac remodeling in the advanced stage of the model , detect cardiac remodeling and cardiac function by immunohistochemistry , and analyze the effects of Nrf2 gene knock - out on the heart in different pathological stages ;
2 . Through the detection of autophagy flow at different time points after TAC operation in WT mice , a TAC model of autophagy dysfunction was established by using the myocardial specificity Atg5 gene knockout mice . The pathological states of the heart and the expression level of Nrf2 and its downstream genes were observed at the time of autophagy dysfunction .
2 . Western Blot was used to detect the effect of autophagy dysfunction on the activation level of each signal pathway when the heart of mice was subjected to pressure load , and compared with the model of Nrf2 knockout mice ' s heart pressure load model , the relationship between them was analyzed .
3 . To establish an animal model of cardiac stress load of Nrf2 / Atg5 double - tapping mice . The relationship between the pathological physiology and the autophagy function of Nrf2 on stress - induced heart failure was detected by immunohistochemistry and western blot . The results showed that the first part : 1 , Nrf2 knockout aggravated the acute myocardial injury induced by TAC operation , but the long - term cardiac remodeling and heart failure caused by operation were relieved .
2 . In the course of heart disease caused by pressure load , the pathological physiology of Nrf2 activation is related to the integrity of autophagy function ;
3 . Over - expression and activation of Nrf2 were present in the mice with autophagy dysfunction . The second part : 1 . The expression of Agt was activated by Nrf2 in the pressure overload heart deficient in autophagy function .
2 . The activation of the TAC - induced Jak / Fyn pathway was blocked by autophagy dysfunction in the heart and autophagy dysfunction induced by pressure overload . Conclusion : 1 . Activation of Nrf2 plays a role in protecting the heart when the autophagy function is complete , and the activation of Nrf2 accelerates the process of cardiac remodeling and cardiac dysfunction when autophagy dysfunction occurs ;
2 . When the autophagy function is complete , the activation of Nrf2 can reduce the myocardial necrosis rate and protect the heart , but when the autophagy function is insufficient , the Nrf2 activates the angiotensin II pathway in the heart to further deteriorate the condition ;
3 . The dysfunction of autophagy resulted in the accumulation of Nrf2 in the nucleus , further the high expression of angiotension , and the accumulation of Nrf2 in the nucleus was related to the blocking of the Jak2 / Fyn signal pathway .

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R54
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本文編號：1859488