【摘要】：研究背景酸堿平衡是維持細(xì)胞內(nèi)環(huán)境穩(wěn)定,保證細(xì)胞正常代謝與功能的前提,而受諸多因素的影響,機(jī)體pH會(huì)發(fā)生一定的波動(dòng)。pH改變會(huì)引發(fā)細(xì)胞內(nèi)一系列信號(hào)變化,參與多種病理生理過(guò)程的調(diào)控。例如嚴(yán)重?zé)齽?chuàng)傷、嚴(yán)重感染、慢性腎衰及代謝性疾病等發(fā)生后,細(xì)胞產(chǎn)酸大于排酸,導(dǎo)致酸中毒,造成細(xì)胞損傷、組織臟器功能障礙。而胞漿堿化與細(xì)胞增殖密切相關(guān),是惡性腫瘤重要特征之一。這提示我們pH對(duì)細(xì)胞功能狀態(tài)具有重要調(diào)控作用。心臟是循環(huán)系統(tǒng)的動(dòng)力器官,嚴(yán)重?zé)齻笤缙诩闯霈F(xiàn)心臟損傷。心臟受損不僅可引起心功能不全,還可進(jìn)一步加重全身其它組織器官缺血缺氧性損害。因此,燒傷后心肌損害的研究具有極其重要的理論與臨床意義。既往研究表明pH下降在介導(dǎo)心臟損傷中發(fā)揮重要作用,酸中毒可導(dǎo)致心肌興奮-收縮耦聯(lián)障礙,造成心肌收縮力減弱,還可直接損害心肌細(xì)胞超微結(jié)構(gòu)造成器質(zhì)性損害。但pH如何引起心肌損傷,其發(fā)生規(guī)律及具體分子機(jī)制仍不完全清楚。自噬是指溶酶體介導(dǎo)的胞漿物質(zhì)被降解循環(huán)再利用的過(guò)程,可更新細(xì)胞內(nèi)衰老、變性或錯(cuò)誤折疊的蛋白,清除多余或受損的細(xì)胞器,以維持細(xì)胞內(nèi)環(huán)境穩(wěn)態(tài)。既往研究證明,自噬在心肌組織中廣泛存在,涉及到許多心臟疾病的心肌病理學(xué)過(guò)程,對(duì)于穩(wěn)定心臟結(jié)構(gòu)和功能有著重要的作用。例如在缺血性心肌病、心力衰竭、心肌炎及心肌肥厚等疾病過(guò)程中,心肌細(xì)胞的自噬活動(dòng)會(huì)增強(qiáng)。但自噬是否參與調(diào)控了pH異常引起心肌損傷及發(fā)揮何種作用目前尚不清楚。有研究表明,細(xì)胞外pH的改變會(huì)影響MCF-10A、MCF-7、Hela等細(xì)胞的自噬活性,Marino等的研究發(fā)現(xiàn)酸性環(huán)境能激活人黑色素瘤細(xì)胞的自噬,從而促進(jìn)細(xì)胞存活,這提示我們pH是影響細(xì)胞自噬重要因素,且具有細(xì)胞特異性。因此我們推測(cè):pH是否通過(guò)調(diào)控自噬引起心肌細(xì)胞功能改變。細(xì)胞自噬的誘導(dǎo)和調(diào)控是一個(gè)非常復(fù)雜而精密的過(guò)程,許多因素均可以誘導(dǎo)細(xì)胞產(chǎn)生自噬,如缺氧、營(yíng)養(yǎng)壓力、氧化應(yīng)激壓力均可激活細(xì)胞自噬,同時(shí)又有許多信號(hào)分子參與自噬的調(diào)控,如能量信號(hào)主要通過(guò)AMPK調(diào)控自噬,絲裂原信號(hào)主要通過(guò)mTORC1影響自噬活性,其他的還有p53、beclin1等信號(hào)通路均可參與自噬的調(diào)控。一磷酸腺苷激活的蛋白激酶(AMP-activated protein kinase,AMPK)是一種進(jìn)化保守的、功能強(qiáng)大的絲/蘇氨酸蛋白激酶,在心臟中,多種損傷如饑餓、缺血缺氧以及氧化應(yīng)激等均可導(dǎo)致AMPK激活�；罨腁MPK在維持細(xì)胞能量代謝和細(xì)胞存活中發(fā)揮重要作用。其中,自噬就是AMPK依賴的一種重要的適應(yīng)和存活機(jī)制。然而關(guān)于AMPK如何調(diào)控自噬,目前存在不同的說(shuō)法。較早的研究認(rèn)為AMPK活化后能夠通過(guò)抑制哺乳動(dòng)物雷帕霉素靶點(diǎn)(Mammalian target of rapamycin,m TOR)活性激活自噬。隨著對(duì)AMPK功能及作用機(jī)制的研究深入,越來(lái)越多的研究發(fā)現(xiàn)AMPK可以直接作用于ULK1調(diào)控自噬,而且大量研究證明AMPK-ULK1是誘導(dǎo)自噬的關(guān)鍵。然而在酸、堿處理?xiàng)l件下AMPK調(diào)控心肌細(xì)胞自噬的下游信號(hào)仍不清楚。本課題旨在研究pH對(duì)心肌細(xì)胞自噬活性的影響及其相關(guān)分子機(jī)制,為pH改變所致的心肌損傷的臨床防治提供新的思路。研究方法1.通過(guò)調(diào)控培養(yǎng)基pH模擬細(xì)胞外酸、堿化條件,采用CCK8及LDH釋放檢測(cè)酸、堿處理?xiàng)l件下心肌細(xì)胞活力的改變。采用蛋白免疫印記(Western blot,WB)和免疫熒光(Immunofluorescence,IF)檢測(cè)酸、堿處理?xiàng)l件下心肌細(xì)胞自噬活性的變化。然后采用熒光分子探針檢測(cè)酸、堿處理?xiàng)l件下溶酶體酸度的改變,進(jìn)一步用巴佛洛霉素A1(bafilomycin A1,Baf A1)抑制溶酶體酸化后,檢測(cè)酸、堿處理?xiàng)l件下自噬活性的變化。2.首先采用WB檢測(cè)酸、堿處理?xiàng)l件下心肌細(xì)胞AMPK活性的變化。使用重組腺病毒轉(zhuǎn)染技術(shù)構(gòu)建了低表達(dá)AMPKα2的心肌細(xì)胞模型。然后干預(yù)AMPK,采用WB和IF檢測(cè)酸、堿處理?xiàng)l件下心肌細(xì)胞自噬水平的變化。此外,采用CCK8、LDH釋放及SYTOX green檢測(cè)酸、堿處理?xiàng)l件下AMPK及其調(diào)控的自噬對(duì)心肌細(xì)胞活力的影響。3.首先采用WB檢測(cè)酸、堿處理?xiàng)l件下心肌細(xì)胞mTORC1活性的變化。干預(yù)AMPK,采用WB檢測(cè)酸、堿處理?xiàng)l件下心肌細(xì)胞mTORC1活性的改變。進(jìn)一步干預(yù)mTORC1,檢測(cè)其對(duì)酸、堿處理?xiàng)l件下心肌細(xì)胞AMPK活性及自噬水平的影響。采用WB檢測(cè)了酸、堿處理?xiàng)l件下ULK1活性的改變。干預(yù)AMPK,檢測(cè)酸、堿處理?xiàng)l件下心肌細(xì)胞ULK1活性的變化。建立了低表達(dá)ULK1的心肌細(xì)胞模型。干預(yù)ULK1,采用WB和IF檢測(cè)酸、堿處理?xiàng)l件下心肌細(xì)胞自噬水平的變化。最后采用CCK8、LDH釋放及SYTOX green染色檢測(cè)酸、堿處理?xiàng)l件下ULK1及其調(diào)控的自噬對(duì)心肌細(xì)胞活力的影響。結(jié)果1.酸化處理3h后,即發(fā)現(xiàn)心肌細(xì)胞活力明顯下降,且隨著處理時(shí)間的延長(zhǎng),心肌細(xì)胞損傷加重;堿化處理后早期,細(xì)胞活力增加,持續(xù)堿化作用可導(dǎo)致細(xì)胞活力下降。酸化處理后,LC3-I向LC3II轉(zhuǎn)換減少;堿化處理誘導(dǎo)LC3-I向LC3II轉(zhuǎn)換增多。酸、堿化處理后,與自噬活性負(fù)相關(guān)的p62蛋白表達(dá)均下降。免疫熒光顯示LC3和LAMP1共定位良好;溶酶體檢測(cè)結(jié)果提示細(xì)胞外pH改變后6 h,溶酶體的酸度未見(jiàn)明顯下降;采用BafA1處理后,酸、堿處理?xiàng)l件下心肌細(xì)胞LC3-II蛋白及p62蛋白均顯著積累。2.酸化處理抑制心肌細(xì)胞AMPK活性,堿化處理誘導(dǎo)心肌細(xì)胞AMPK激活。酸化處理6 h后,細(xì)胞自噬水平下降,而使用Met激活A(yù)MPK后細(xì)胞自噬水平升高,使用AMPKα2 sh RNA抑制AMPK活性可逆轉(zhuǎn)Met誘導(dǎo)的自噬活性上調(diào);堿化處理后6h自噬水平升高,使用AMPKα2 sh RNA抑制AMPK活性可顯著阻斷堿化處理誘導(dǎo)的自噬水平上調(diào)。酸化處理6 h后,心肌細(xì)胞活力下降,死細(xì)胞數(shù)目增加,而使用Met激活A(yù)MPK和自噬活性后,酸化處理誘導(dǎo)的細(xì)胞損傷減輕,死細(xì)胞數(shù)目減少,使用AMPKα2 sh RNA抑制AMPK活性可阻斷這一效應(yīng);堿化處理6 h后,細(xì)胞活力升高,死亡細(xì)胞減少,使用AMPKα2 sh RNA抑制AMPK活性后細(xì)胞活力下降,細(xì)胞死亡數(shù)目增多。3.酸化處理下調(diào)心肌細(xì)胞mTORC1活性,堿化處理誘導(dǎo)mTORC1激活。酸、堿處理?xiàng)l件下,使用Met和AMPKα2 sh RNA激活/抑制AMPK6 h后,mTORC1活性無(wú)明顯變化。酸、堿處理?xiàng)l件下,使用m EGF/RAPA激活/抑制mTORC16 h后,AMPK活性和自噬水平均無(wú)顯著變化。酸化處理抑制心肌細(xì)胞ULK1活性,堿化處理誘導(dǎo)ULK1激活。酸化處理6 h后,ULK1活性下降,而使用Met激活A(yù)MPK后細(xì)胞ULK1活性增強(qiáng),使用AMPKα2 sh RNA抑制AMPK活性可逆轉(zhuǎn)Met誘導(dǎo)的ULK1激活;堿化處理上調(diào)ULK1活性,使用AMPKα2 sh RNA干擾后可抑制這一效應(yīng)。酸化處理6 h后,自噬水平下降,而使用Met激活A(yù)MPK和ULK1后細(xì)胞自噬水平上調(diào),進(jìn)一步使用ULK1 sh RNA抑制ULK1活性可逆轉(zhuǎn)Met誘導(dǎo)的自噬活性增高;堿化處理誘導(dǎo)自噬水平升高,使用ULK1 sh RNA敲降ULK1表達(dá)可顯著阻斷這一過(guò)程。酸化處理6h后,細(xì)胞活力下降,死亡細(xì)胞增加,而使用Met激活A(yù)MPK和自噬活性可減輕酸化誘導(dǎo)的細(xì)胞損傷,進(jìn)一步使用ULK1 sh RNA敲降ULK1表達(dá)可阻斷這一效應(yīng);堿化處理6 h后,細(xì)胞活力升高,死亡細(xì)胞減少,使用ULK1 sh RNA敲降ULK1表達(dá)后細(xì)胞活力下降,細(xì)胞死亡數(shù)目增多。結(jié)論1.酸化處理導(dǎo)致細(xì)胞損活力下降,堿化處理后早期細(xì)胞活力升高,持續(xù)作用細(xì)胞活力下降。酸化處理后,心肌細(xì)胞自噬活性明顯下降;堿化處理誘導(dǎo)自噬激活,酸、堿化處理后均不伴有自噬通量的損傷。2.pH可調(diào)控心肌細(xì)胞AMPK的活性,AMPK介導(dǎo)了pH對(duì)原代心肌細(xì)胞自噬活性及細(xì)胞活力的調(diào)控。3.pH可調(diào)控心肌細(xì)胞中mTORC1的活性,酸、堿處理?xiàng)l件下,AMPK和mTORC1的相互作用不明顯,mTORC1信號(hào)通路在pH介導(dǎo)的心肌細(xì)胞自噬調(diào)控中不發(fā)揮主要作用。酸、堿處理?xiàng)l件下,ULK1可能是AMPK的重要下游靶標(biāo)之一,AMPK可能通過(guò)調(diào)控ULK1信號(hào)通路介導(dǎo)心肌細(xì)胞自噬活性和細(xì)胞活力的改變。4.酸、堿處理?xiàng)l件下,早期發(fā)生的細(xì)胞自噬均有保護(hù)心肌的作用,抑制自噬活化會(huì)加重心肌細(xì)胞損傷。5.在嚴(yán)重?zé)齻剐菘酥委熤?如何使血液酸堿度控制在既不影響血紅蛋白釋放氧,又不影響細(xì)胞生命活動(dòng)的范圍內(nèi),對(duì)減輕組織細(xì)胞缺血缺氧損害,提高抗休克治療效果具有非常重要的臨床指導(dǎo)意義。6.本課題在一定程度上闡明了pH對(duì)心肌細(xì)胞自噬的影響及其相關(guān)機(jī)制,為pH改變所致的心肌損傷的臨床防治提供新的思路。
[Abstract]:BACKGROUND Acid-base balance is the premise of maintaining the stability of intracellular environment and ensuring the normal metabolism and function of cells. Influenced by many factors, the body pH will fluctuate. pH changes can cause a series of intracellular signal changes and participate in the regulation of various pathophysiological processes, such as severe burns, severe infection, chronic renal failure and so on. After the occurrence of metabolic diseases, cells produce more acid than they expel it, which leads to acidosis, cell damage and organ dysfunction. Cytoplasmic alkalization is closely related to cell proliferation, which is one of the important characteristics of malignant tumors. This suggests that pH plays an important role in regulating cell function. Cardiac injury occurs early after injury. Cardiac injury can not only cause cardiac insufficiency, but also aggravate ischemia-hypoxia damage in other tissues and organs of the whole body. Therefore, the study of myocardial damage after burns has extremely important theoretical and clinical significance. Toxicity can lead to disturbance of excitation-contraction coupling, weaken the contractility of myocardium, and directly damage the ultrastructure of myocardial cells, resulting in organic damage. Autophagy has been proven to be widespread in myocardial tissues and involved in myocardial pathological processes in many heart diseases. It plays an important role in stabilizing cardiac structure and function, such as ischemia. It is not clear whether autophagy is involved in the regulation of myocardial injury caused by abnormal pH and what role it plays. Studies have shown that changes in extracellular pH can affect the autophagy of MCF-10A, MCF-7, Hela and Marino. It was found that acidic environment can activate autophagy of human melanoma cells and promote cell survival. This suggests that pH is an important factor affecting cell autophagy and has cell specificity. Many factors can induce autophagy, such as hypoxia, nutritional stress and oxidative stress. At the same time, many signal molecules participate in the regulation of autophagy. For example, energy signal mainly regulates autophagy through AMPK, mitogen signal mainly affects autophagy activity through mTORC1, and other factors include p53, becl. AMP-activated protein kinase (AMPK) is an evolutionarily conserved and powerful serine/threonine protein kinase. In the heart, a variety of damage such as starvation, ischemia and hypoxia, and oxidative stress can lead to the activation of AMPK. Autophagy is an important adaptation and survival mechanism of AMPK dependence. However, there are different opinions about how AMPK regulates autophagy. Earlier studies suggested that AMPK activation can inhibit mammalian target of rapamycin (m). TOR) Activated autophagy. With the further study of the function and mechanism of AMPK, more and more studies have found that AMPK can directly affect ULK1 to regulate autophagy, and a large number of studies have proved that AMPK-ULK1 is the key to induce autophagy. However, the downstream signal of AMPK regulating cardiomyocyte autophagy under acid and alkali treatment is still unclear. To study the effects of pH on autophagy of cardiomyocytes and its related molecular mechanisms, and to provide new ideas for the clinical prevention and treatment of myocardial injury induced by pH changes. Methods 1. The extracellular acidification and alkalization conditions were simulated by adjusting the pH of the medium, and the changes of cardiomyocyte viability were detected by CCK8 and LDH release. Western blot (WB) and immunofluorescence (IF) were used to detect the changes of autophagy activity of cardiac myocytes under acid and alkali treatment. Then the changes of lysosomal acidity were detected by fluorescent molecular probe under acid and alkali treatment. After inhibition of lysosomal acidification by bafilomycin A1 (Baf A1), the acid was detected. The changes of autophagy activity under alkali treatment were studied. 2. The changes of AMPK activity in cardiomyocytes under acid and alkali treatment were detected by WB. A myocardial cell model with low expression of AMPK alpha2 was constructed by recombinant adenovirus transfection technique. CCK8, LDH release and SYTOX green were used to detect the effect of AMPK and its regulated autophagy on myocardial cell viability under alkali and acid treatments. WB was used to detect the changes of ULK1 activity. Intervention of AMPK was performed to detect the changes of ULK1 activity in cardiomyocytes under acid and alkali treatment. A myocardial cell model with low expression of ULK1 was established. Intervention of ULK1 with WB and IF was used to detect the changes of ULK1 activity in cardiomyocytes under acid and alkali treatment. The changes of autophagy level of cardiomyocytes were detected by CCK8, LDH release and SYTOX green staining. The effect of ULK1 and its autophagy on the viability of cardiomyocytes was detected under alkaline treatment. Results 1. After 3 hours of acidification treatment, the viability of cardiomyocytes decreased significantly, and the damage of cardiomyocytes was aggravated with the prolongation of treatment time. After acidification, the conversion of LC3-I to LC3II was decreased; after alkalization, the conversion of LC3-I to LC3II was increased; after acidification, the expression of p62 protein negatively correlated with autophagic activity was decreased. Immunofluorescence showed that LC3 and LAMP1 were well co-located; lysosome assay showed that the expression of Lysozyme was positive. The results showed that the acidity of lysosome did not decrease significantly 6 hours after the change of extracellular pH, and the accumulation of LC3-II protein and p62 protein in cardiac myocytes under acid and alkaline treatment was significant after BafA1 treatment. 2. Acidification inhibited the activity of AMPK in cardiac myocytes, alkaline treatment induced the activation of AMPK in cardiac myocytes. The autophagy level of AMPK activated by Met increased, and the inhibition of AMPK activity by AMPK alpha 2sh RNA reversed the up-regulation of autophagy induced by Met; the increase of autophagy level 6 h after alkalinization treatment, and the inhibition of AMPK activity by AMPK alpha 2sh RNA significantly blocked the up-regulation of autophagy induced by alkalinization treatment. The number of AMPK cells increased, but the number of dead cells decreased and the damage of AMPK cells induced by acidification was alleviated after activation of AMPK and autophagy by Met. Inhibition of AMPK activity by AMPK alpha 2sh RNA blocked this effect. After 6 hours of alkalinization treatment, the cell viability increased and the dead cells decreased. After inhibition of AMPK activity by AMPK alpha 2sh RNA, the cell viability decreased. Acidification decreased the activity of mTORC1 and alkalinization induced the activation of mTORC1. Under acid and alkaline treatment, the activity of mTORC1 did not change significantly after activation/inhibition of AMPK by Met and AMPK alpha2 sh RNA for 6 h. Under acid and alkaline treatment, the activity of AMPK and the level of autophagy did not change significantly after activation/inhibition of mTORC16 h by m EGF/RAPA. Acidification treatment inhibited ULK1 activity in cardiomyocytes and alkalinization treatment induced ULK1 activation. After 6 hours of acidification treatment, ULK1 activity decreased, while after activation of AMPK by Met, ULK1 activity increased. Inhibition of AMPK activity by AMPK alpha 2 sh RNA reversed Met-induced ULK1 activation; alkalinization treatment increased ULK1 activity, and after AMPK alpha 2 sh RNA interference, ULK1 activity was inhibited. After 6 h of acidification, the autophagy level decreased, while the autophagy level increased after the activation of AMPK and ULK1 by Met. Further inhibition of ULK1 activity by ULK1 sh RNA reversed the increase of autophagy induced by Met, and the increase of autophagy level induced by alkalinization was significantly blocked by knockdown of ULK1 expression by ULK1 sh RNA. After 6 hours of treatment, the cell viability decreased and the dead cells increased, while the activation of AMPK and autophagy by Met alleviated acidification-induced cell injury, which was blocked by ULK1 sh RNA knockdown. After 6 hours of alkalinization treatment, the cell viability increased and the dead cells decreased. After knockdown of ULK1 expression by ULK1 sh RNA, the cell viability decreased. The number of cell death increased. Conclusion 1. Acidification decreased the activity of cell damage, increased the activity of cell in the early stage of alkalization, and decreased the activity of sustained acting cells. AMPK and AMPK mediated the regulation of pH on autophagy and viability of primary cardiomyocytes. 3. pH regulated the activity of mTORC1 in cardiomyocytes. Under acid and alkali treatment, the interaction between AMPK and mTORC1 was not obvious. The mTORC1 signaling pathway did not play a major role in pH-mediated autophagy of cardiomyocytes. ULK1 may be one of the important downstream targets of AMPK. AMPK may mediate the changes of autophagy and cell viability by regulating ULK1 signaling pathway. 4. Under acid and alkali treatment, early autophagy can protect myocardium, and inhibition of autophagy may aggravate myocardial injury. 5. In severe burns, AMPK can resist shock. In the treatment, how to control the blood acidity and alkalinity in the range of not affecting hemoglobin to release oxygen, but also not affecting cell life activity, has very important clinical significance to reduce the damage of tissue and cell ischemia and hypoxia, improve the therapeutic effect of anti-shock. 6. To some extent, this topic clarifies the effect of pH on myocardial autophagy. And its related mechanisms will provide new ideas for clinical prevention and treatment of myocardial injury induced by pH changes.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R54

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相關(guān)期刊論文 前1條

1 姚偉,錢桂生,楊曉靜;NHE-1與大鼠肺動(dòng)脈平滑肌細(xì)胞增殖和凋亡(英文)[J];Chinese Medical Journal;2002年01期

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本文編號(hào)：2206918