本文選題：自噬 + 系膜細(xì)胞　； 參考：《第二軍醫(yī)大學(xué)》2014年博士論文

【摘要】：研究背景和目的 慢性腎臟�。╟hronic kidney disease CKD）是全球范圍內(nèi)越來越嚴(yán)重的公共衛(wèi)生問題。CKD患者大多數(shù)最終不得不接受腎臟替代治療，并且持續(xù)占用大量的醫(yī)療資源。在CKD的治療中，延緩腎功能持續(xù)進(jìn)展是臨床與科研工作者關(guān)注的重點(diǎn)。在腎臟固有細(xì)胞中，腎小球系膜細(xì)胞（mesangial cell MC）在CKD進(jìn)展中的地位和作用越來越受到人們的重視。系膜細(xì)胞在不同的損傷因素下，可能表現(xiàn)出增殖肥大、凋亡、裂解、遷移、產(chǎn)生過量的系膜基質(zhì)、產(chǎn)生過量的活性氧自由基（ROS）等等，造成腎損害加重及CKD持續(xù)的進(jìn)展。所以，深入研究系膜細(xì)胞的生理功能以及在損傷條件下細(xì)胞生理功能的變化，有助于進(jìn)一步了解系膜細(xì)胞的損傷應(yīng)激系統(tǒng)，從而為監(jiān)測(cè)與治療CKD的進(jìn)展提供更好的視角及切入點(diǎn)。 自噬是體內(nèi)最為基本的生理過程之一，真核細(xì)胞自噬活性的變化廣泛參與了體內(nèi)炎癥、增殖、凋亡等復(fù)雜的生理及病理過程，并在其中發(fā)揮著極為重要甚至是決定性的作用。已有研究表明自噬的失調(diào)與腫瘤、神經(jīng)退行性疾病、心血管疾病以及感染等疾病密切相關(guān)。而自噬在腎臟疾病中的作用正受到越來越多的關(guān)注。自噬活性的變化與腎小球系膜細(xì)胞應(yīng)激損傷之間的關(guān)系現(xiàn)只有很少的研究報(bào)道。自噬是否參與了腎小球系膜細(xì)胞的損傷應(yīng)激，這種損傷應(yīng)激在CKD的進(jìn)展中作用如何，值得進(jìn)一步深入的研究。最新的研究表明，自噬的激活可以增加系膜細(xì)胞內(nèi)I型膠原的降解，從而減少腎小球系膜基質(zhì)的增多及抑制腎臟纖維化的進(jìn)展。還有研究表明，引起腎臟纖維化最為重要的分子—TGF-β，能夠誘導(dǎo)系膜細(xì)胞的自噬水平出現(xiàn)變化，影響著下游相關(guān)凋亡相關(guān)caspase-3分子的水平變化�？梢�，系膜細(xì)胞的自噬活性的變化參與了CKD進(jìn)展中系膜細(xì)胞向成纖維細(xì)胞表型的轉(zhuǎn)化，并可能在其中發(fā)揮了極為重要的作用。 腎素-血管緊張素-醛固酮系統(tǒng)（RASS）的異常激活在慢性腎臟病的發(fā)生及發(fā)展進(jìn)程中具有極為重要的意義。而近年來，醛固酮作為CKD的重要獨(dú)立損傷因子，越來越受到人們的關(guān)注。在腎臟的炎癥狀態(tài)及纖維化發(fā)展過程中醛固酮發(fā)揮重要作用，影響著系膜細(xì)胞及其他腎臟固有細(xì)胞。國(guó)外研究結(jié)果表明，醛固酮通過激活活性氧（ROS）及表皮生長(zhǎng)因子受體（EGFR），通過RAS/MAPK和PI3K/Akt信號(hào)通路誘使腎小球系膜細(xì)胞增殖。增殖的腎小球系膜細(xì)胞分泌系膜基質(zhì)增加，并向纖維化的表型轉(zhuǎn)化，促進(jìn)了腎臟間質(zhì)細(xì)胞的纖維化及CKD的進(jìn)展。 我們注意到，作為來自循環(huán)的系膜細(xì)胞損傷因子——醛固酮及維持系膜細(xì)胞自穩(wěn)態(tài)及增加系膜基質(zhì)清除的生理過程——自噬，都參與了系膜細(xì)胞的損傷應(yīng)激及纖維化表型轉(zhuǎn)化過程。醛固酮可以促進(jìn)這一過程，系膜細(xì)胞自噬激活可以抑制這種轉(zhuǎn)化。還有動(dòng)物實(shí)驗(yàn)證實(shí)，醛固酮可以造成腎臟細(xì)胞的衰老，而自噬正是體內(nèi)細(xì)胞對(duì)抗衰老的主要生理過程。綜合上述的研究進(jìn)展，醛固酮可能通過某些途徑對(duì)系膜細(xì)胞的自噬產(chǎn)生負(fù)性調(diào)控，而這種自噬活性的變化可能參與了系膜細(xì)胞的損傷應(yīng)激及CKD的進(jìn)展過程。 通過本研究，我們想進(jìn)一步明確醛固酮對(duì)系膜細(xì)胞自噬的影響，初步的探討醛固酮對(duì)自噬的調(diào)控作用產(chǎn)生的生理及病理意義，積極探索可能參與其中的信號(hào)通路及分子機(jī)制。細(xì)胞的自噬往往與線粒體功能的變化及細(xì)胞內(nèi)ROS的水平具有很大的關(guān)聯(lián)，而血管緊張素II及醛固酮都參與了系膜細(xì)胞線粒體的損傷及ROS的激活過程，我們也試圖進(jìn)一步研究二者與系膜細(xì)胞線粒體損傷的關(guān)系。 實(shí)驗(yàn)方法 1．使用CCK8方法及流式細(xì)胞術(shù)測(cè)定醛固酮及血管緊張素對(duì)于系膜細(xì)胞增殖的影響。 2．通過western blot方法檢測(cè)在饑餓、醛固酮、血管緊張素、表皮生長(zhǎng)因子等不同干預(yù)的條件下系膜細(xì)胞自噬相關(guān)的蛋白標(biāo)志物L(fēng)C3、P62/SQSTM1的表達(dá)水平變化。檢測(cè)信號(hào)通路蛋白EGFR、ERK、GAB1、PARP及剪切體PARP、AMPK及Beclin-1等蛋白表達(dá)水平的變化。 3.光鏡下觀察H2O2刺激系膜細(xì)胞在有無醛固酮長(zhǎng)期干預(yù)下的凋亡變化。 4．通過向系膜細(xì)胞轉(zhuǎn)入GFP-LC3的真核表達(dá)質(zhì)粒，用激光共聚焦顯微鏡觀察不同的刺激影響下系膜細(xì)胞熒光自噬點(diǎn)數(shù)量的變化并進(jìn)行統(tǒng)計(jì)學(xué)分析。 5.通過電子顯微鏡觀察不同的干預(yù)條件下系膜細(xì)胞自噬泡數(shù)量的變化，并進(jìn)行統(tǒng)計(jì)學(xué)分析。 6.使用Mito-TrackerGreen探針（Invitrogen公司）檢測(cè)不同的干預(yù)條件下系膜細(xì)胞線粒體的數(shù)量變化。使用Mito-ID細(xì)胞外液酸度檢測(cè)試劑盒檢測(cè)不同干預(yù)條件下細(xì)胞外液酸度變化情況，顯示線粒體損傷后細(xì)胞糖酵解水平的變化。 7．免疫共沉淀的方法檢測(cè)醛固酮干預(yù)對(duì)于Beclin-1-BCL2復(fù)合體的影響。 8. Real-timePCR方法檢測(cè)了自噬相關(guān)基因及線粒體相關(guān)的基因在醛固酮及血管緊張素干預(yù)下的表達(dá)情況。 結(jié)果 1.在體外培養(yǎng)的腎小球系膜細(xì)胞系HMCL及RMC中，，高于正常濃度的醛固酮與血管緊張素II不能使兩種細(xì)胞系的增殖水平出現(xiàn)明顯的變化。流式細(xì)胞儀檢測(cè)干預(yù)后G2/M期細(xì)胞的比率沒有明顯的升高。 2.醛固酮可以抑制血清饑餓誘導(dǎo)的系膜細(xì)胞自噬活性的升高，且這種抑制表現(xiàn)為劑量依賴性。western blot檢測(cè)發(fā)現(xiàn)醛固酮干預(yù)后EBSS培養(yǎng)的系膜細(xì)胞LC3II表達(dá)降低及P62/SQSTM1表達(dá)升高。共聚焦顯微鏡觀察發(fā)現(xiàn)饑餓誘導(dǎo)的系膜細(xì)胞HMCL自噬點(diǎn)數(shù)量增加，而醛固酮可以抑制這種效應(yīng)。電鏡下自噬泡的的計(jì)數(shù)觀察同樣證實(shí)醛固酮可以抑制饑餓誘導(dǎo)的系膜細(xì)胞自噬活化。 3.通過western blot、熒光顯微鏡觀察自噬點(diǎn)的形成以及電鏡結(jié)果均可證實(shí)雷帕霉素可以激活腎小球系膜細(xì)胞的自噬，醛固酮不能夠有效抑制這種自噬的激活效應(yīng)。 4.在醛固酮導(dǎo)致的長(zhǎng)期的自噬抑制狀態(tài)下，系膜細(xì)胞在面臨氧化應(yīng)激時(shí)更容易發(fā)生凋亡。表現(xiàn)為在相同的時(shí)間點(diǎn)，凋亡細(xì)胞的比率明顯增加，western blot檢測(cè)發(fā)現(xiàn)PARP蛋白的剪切體的表達(dá)上升。 5.醛固酮、血管緊張素II能夠在5、15、60分鐘內(nèi)輕度激活EGFR，促使其磷酸化的水平提高，但這種激活作用遠(yuǎn)遠(yuǎn)弱于EGF的直接刺激，三者激活EGFR的效應(yīng)按強(qiáng)度大小排列為EGFAngIIAld。但EGF刺激EGFR磷酸化的強(qiáng)度隨時(shí)間很快衰減，而血管緊張素II與醛固酮對(duì)EGFR磷酸化則在1小時(shí)內(nèi)緩慢增強(qiáng)，至48小時(shí)仍有持續(xù)的EGFR磷酸化及ERK的磷酸化。提示細(xì)胞在高濃度的醛固酮及血管緊張素II持續(xù)刺激下可表現(xiàn)為EGFR及ERK的持續(xù)激活狀態(tài)，且同時(shí)具有時(shí)間依賴性和劑量依賴性。 6.醛固酮可以降低在饑餓條件下AMPK的磷酸化水平，通過降低AMPK的磷酸化來抑制Beclin-1在Ser93/96位點(diǎn)的磷酸化，進(jìn)而增加了其與BCL2的結(jié)合，導(dǎo)致Beclin-1與VPS34復(fù)合體結(jié)合減少，而抑制自噬。 7.與對(duì)照組及血清饑餓組相比，醛固酮干預(yù)24小時(shí)對(duì)于系膜細(xì)胞線粒體的數(shù)量有明顯的增加，血管緊張素II效果更為顯著。血管緊張素II不僅可以增加線粒體的數(shù)量，更影響了線粒體的形態(tài)，熒光顯微鏡下可見大量線粒體融合成拉絲狀，電鏡下線粒體腫脹，寬大畸形，部分融合，線粒體嵴排列紊亂。血管緊張素II及醛固酮均能降低線粒體的儲(chǔ)備功能，削弱系膜細(xì)胞線粒體對(duì)于應(yīng)激的反應(yīng)性，但血管緊張素II對(duì)其影響更大。 8.醛固酮及血管緊張素II均可以造成部分自噬相關(guān)基因的表達(dá)發(fā)生變化，其中血管緊張素II多造成這些基因表達(dá)的下調(diào)，而醛固酮?jiǎng)t造成這些基因的表達(dá)上調(diào)。醛固酮對(duì)于部分線粒體相關(guān)的基因的表達(dá)影響較輕，而血管緊張素II的影響較明顯。特別是血管緊張素II對(duì)線粒體相關(guān)基因的影響程度大于醛固酮。結(jié)論 1.本實(shí)驗(yàn)第一次發(fā)現(xiàn)了醛固酮對(duì)于系膜細(xì)胞饑餓誘導(dǎo)的自噬激活的抑制效應(yīng)。這種抑制效應(yīng)參與了醛固酮在氧化應(yīng)激中對(duì)系膜細(xì)胞的協(xié)同損傷。 2.醛固酮抑制自噬效應(yīng)的靶點(diǎn)可能在雷帕霉素的上游。有可能是通過抑制AMPK磷酸化及下游的Beclin-1Ser93/96位點(diǎn)磷酸化。 3.血管緊張素II及醛固酮都能不同程度的損傷系膜細(xì)胞線粒體，激活ROS及造成EGFR的持續(xù)激活，前者較后者更為嚴(yán)重。
[Abstract]:Background and purpose of research
Chronic kidney disease (chronic kidney disease CKD) is an increasingly serious public health problem worldwide. Most of the patients with.CKD have to accept renal replacement therapy and continue to occupy a large number of medical resources. In the treatment of CKD, the continued progress of renal function is the focus of attention of clinical and scientific researchers. In the cells, the role and role of mesangial cell MC in the progression of CKD has been paid more and more attention. Under different damage factors, mesangial cells may show proliferation, apoptosis, cracking, migration, excessive mesangial matrix, excessive reactive oxygen free radical (ROS) and so on, resulting in renal damage plus renal damage. Therefore, it is important to further study the physiological functions of the mesangial cells and the changes in the physiological functions of the cells under the condition of damage. It is helpful to further understand the damage stress system of mesangial cells, and thus provide a better perspective and breakthrough point for the monitoring and treatment of the progress of CKD.
Autophagy is one of the most basic physiological processes in the body. The changes in autophagic activity of eukaryotic cells are widely involved in the complex physiological and pathological processes, such as inflammation, proliferation, apoptosis and other complex processes in the body, and play a very important and even decisive role in it. The role of autophagy in renal diseases is becoming more and more concerned. The relationship between the changes of autophagy and the stress damage of glomerular mesangial cells is only rarely reported. Is autophagy involved in the damage stress of glomerular mesangial cells; this damage stress is progresses in the progress of CKD The latest research shows that the activation of autophagy can increase the degradation of I type collagen in mesangial cells, thus reducing the increase of mesangial matrix and the progress in inhibiting renal fibrosis. And the study shows that the most important molecule TGF- beta, which causes renal fibrosis, can induce mesangial cells. The change of autophagy affects the level of Caspase-3 molecules associated with apoptosis in the downstream. It is seen that the changes in autophagy of mesangial cells are involved in the transformation of mesangial cells to the phenotype of fibroblasts in the progress of CKD, and may play an important role in it.
The abnormal activation of the renin angiotensin aldosterone system (RASS) is of great importance in the development and development of chronic renal disease. In recent years, aldosterone has attracted more and more attention as an important independent damage factor of CKD. Aldosterone plays an important role in the inflammatory state of the kidney and the development of fibrosis. The results showed that aldosterone induced the proliferation of glomerular mesangial cells by activating reactive oxygen species (ROS) and epidermal growth factor receptor (EGFR) by activating the active oxygen (ROS) and epidermal growth factor receptor (EGFR). The transformation promoted the fibrosis of renal interstitial cells and the progress of CKD.
It is noted that aldosterone as a damage factor from the circulatory mesangial cells, the self homeostasis of aldosterone and the maintenance of mesangial cells, and the physiological process of increasing the removal of the mesangial matrix - autophagy, is involved in the damage stress and phenotypic transformation of the mesangial cells. Aldosterone can promote this process and the autophagy activation of mesangial cells can be suppressed. And animal experiments have proved that aldosterone can cause the aging of renal cells, and autophagy is the main physiological process of cell aging in vivo. Synthesis of aldosterone may be a negative regulation of autophagy in mesangial cells through some ways, and the changes in autophagy may be involved in the system. Damage stress of membrane cells and the progress of CKD.
Through this study, we want to further clarify the effect of aldosterone on autophagy in mesangial cells, preliminarily explore the physiological and pathological significance of aldosterone in the regulation of autophagy, and actively explore the signaling pathways and molecular mechanisms involved in it. The autophagy of the cells is often associated with the changes in the function of the grain body and the level of ROS in the cell. The relationship between angiotensin II and aldosterone is involved in the mitochondrial damage of mesangial cells and the activation of ROS. We also try to further study the relationship between the two and the mitochondrial damage in mesangial cells.
Experimental method
1. the effects of aldosterone and angiotensin on proliferation of mesangial cells were measured by CCK8 and flow cytometry.
2. Western blot method was used to detect the changes in the expression level of the autophagy related protein markers, LC3, P62/SQSTM1, in the conditions of starvation, aldosterone, angiotensin, epidermal growth factor, etc., and to detect the changes in the expression level of the protein EGFR, ERK, GAB1, PARP and the PARP, AMPK and Beclin-1 protein of the signaling pathway protein, ERK, GAB1, PARP and shear.
3. the apoptosis of mesangial cells stimulated by H2O2 was observed under light microscope.
4. the changes in the number of autophagic points of the mesangial cells under the influence of different stimuli were observed and analyzed by a laser confocal microscope through the eukaryotic expression plasmid transferred into the mesangial cells to GFP-LC3.
5. the number of autophagic vacuoles in mesangial cells under different intervention conditions was observed by electron microscopy and analyzed statistically.
6. Mito-TrackerGreen probe (Invitrogen) was used to detect the changes in the number of mitochondria in mesangial cells under different intervention conditions. Mito-ID extracellular acidity detection kit was used to detect the change of acidity of extracellular fluid under different intervention conditions, and the changes of the level of fine cell glycolysis after mitochondrial injury were shown.
7. co immunoprecipitation was used to detect the effect of aldosterone intervention on Beclin-1-BCL2 complex.
8. the expression of autophagy related genes and mitochondrial associated genes under aldosterone and angiotensin were detected by Real-timePCR.
Result
1. in cultured glomerular mesangial cell lines HMCL and RMC, higher levels of aldosterone and angiotensin II were not significantly higher than normal concentrations of aldosterone and angiotensin, and the rate of G2/M cells was not significantly increased after flow cytometry.
2. aldosterone could inhibit the increase of autophagy in the mesangial cells induced by serum starvation, and this inhibition showed that the dose dependent.Western blot detection was used to detect the decrease of LC3II expression and the increase of P62/SQSTM1 expression in the mesangial cells cultured by EBSS. The confocal microscope observed the autophagy point of HMCL in the mesangial cells induced by starvation. The number of aldosterone could inhibit the effect. The observation of autophagic vesicles under electron microscope also confirmed that aldosterone could inhibit the activation of autophagy in the mesangial cells induced by starvation.
3. by Western blot, the formation of autophagic points and the results of electron microscopy can prove that rapamycin can activate autophagy in glomerular mesangial cells. Aldosterone can not effectively inhibit the activation of this autophagy.
4. in the state of long-term autophagy induced by aldosterone, mesangial cells are more prone to apoptosis in the face of oxidative stress. The percentage of apoptotic cells increased significantly at the same time point, and the expression of the shear body of PARP protein increased by Western blot detection.
5. aldosterone, angiotensin II, can activate EGFR slightly within 5,15,60 minutes to increase the level of phosphorylation, but this activation is far weaker than the direct stimulation of EGF. The effect of the three activates EGFR on the intensity of EGFAngIIAld. but EGF stimulates EGFR phosphorylation rapidly with time, and angiotensin II and Aldosterone was enhanced slowly for EGFR phosphorylation in 1 hours, and continued EGFR phosphorylation and phosphorylation of ERK to 48 hours. It was suggested that the cells exhibited persistent activation of EGFR and ERK at high concentrations of aldosterone and angiotensin II, and were dependent on the time dependent and dose-dependent manner.
6. aldosterone can reduce the phosphorylation level of AMPK under starvation, inhibit phosphorylation of AMPK to inhibit the phosphorylation of Beclin-1 at the Ser93/96 site, and then increase its binding with BCL2, resulting in a decrease in the binding of Beclin-1 to the VPS34 complex and inhibition of autophagy.
7. compared with the control group and the serum starvation group, the number of mitochondria in the mesangial cells increased significantly for 24 hours, and the effect of angiotensin II was more significant. Angiotensin II could not only increase the number of mitochondria, but also affect the morphology of mitochondria. Mitochondrial swelling, broad deformities, partial fusion and mitochondrial crista disorder. Angiotensin II and aldosterone both reduce the mitochondrial reserve function and weaken the response to stress in the mesangial cell mitochondria, but angiotensin II has a greater impact on it.
8. aldosterone and angiotensin II can cause changes in the expression of partial autophagy related genes, in which angiotensin II causes the downregulation of these gene expressions, and aldosterone causes the up-regulated expression of these genes. Aldosterone has a light influence on the expression of some mitochondrial related genes, and the effect of angiotensin II The effect of angiotensin II on mitochondrial related genes was greater than that of aldosterone.
1. the inhibition effect of aldosterone on the activation of autophagy induced by the starvation of mesangial cells was first discovered in this experiment. This inhibitory effect was involved in the synergistic damage of aldosterone to mesangial cells during oxidative stress.
2. aldosterone may inhibit the autophagy effect upstream of rapamycin, probably by inhibiting AMPK phosphorylation and downstream Beclin-1Ser93/96 site phosphorylation.
3. angiotensin II and aldosterone can damage the mitochondria of mesangial cells in different degrees, activate ROS and cause EGFR to continue to activate. The former is more serious than the latter.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R692

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 賴凌云,顧勇,陳靖,郁勝?gòu)?qiáng),馬驥,楊海春,林善錟;大鼠系膜細(xì)胞醛固酮的合成及其對(duì)細(xì)胞外基質(zhì)生成的影響[J];中華醫(yī)學(xué)雜志;2003年21期

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