本文選題：自噬 + 系膜細胞�。� 參考：《第二軍醫(yī)大學》2014年博士論文

【摘要】：研究背景和目的 慢性腎臟�。╟hronic kidney disease CKD）是全球范圍內越來越嚴重的公共衛(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)進展是臨床與科研工作者關注的重點。在腎臟固有細胞中，腎小球系膜細胞（mesangial cell MC）在CKD進展中的地位和作用越來越受到人們的重視。系膜細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、產生過量的系膜基質、產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)的進展。所以，深入研究系膜細胞的生理功能以及在損傷條件下細胞生理功能的變化，有助于進一步了解系膜細胞的損傷應激系統(tǒng)，從而為監(jiān)測與治療CKD的進展提供更好的視角及切入點。 自噬是體內最為基本的生理過程之一，真核細胞自噬活性的變化廣泛參與了體內炎癥、增殖、凋亡等復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與腫瘤、神經退行性疾病、心血管疾病以及感染等疾病密切相關。而自噬在腎臟疾病中的作用正受到越來越多的關注。自噬活性的變化與腎小球系膜細胞應激損傷之間的關系現(xiàn)只有很少的研究報道。自噬是否參與了腎小球系膜細胞的損傷應激，這種損傷應激在CKD的進展中作用如何，值得進一步深入的研究。最新的研究表明，自噬的激活可以增加系膜細胞內I型膠原的降解，從而減少腎小球系膜基質的增多及抑制腎臟纖維化的進展。還有研究表明，引起腎臟纖維化最為重要的分子—TGF-β，能夠誘導系膜細胞的自噬水平出現(xiàn)變化，影響著下游相關凋亡相關caspase-3分子的水平變化。可見，系膜細胞的自噬活性的變化參與了CKD進展中系膜細胞向成纖維細胞表型的轉化，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的重要獨立損傷因子，越來越受到人們的關注。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，影響著系膜細胞及其他腎臟固有細胞。國外研究結果表明，醛固酮通過激活活性氧（ROS）及表皮生長因子受體（EGFR），通過RAS/MAPK和PI3K/Akt信號通路誘使腎小球系膜細胞增殖。增殖的腎小球系膜細胞分泌系膜基質增加，并向纖維化的表型轉化，促進了腎臟間質細胞的纖維化及CKD的進展。 我們注意到，作為來自循環(huán)的系膜細胞損傷因子——醛固酮及維持系膜細胞自穩(wěn)態(tài)及增加系膜基質清除的生理過程——自噬，都參與了系膜細胞的損傷應激及纖維化表型轉化過程。醛固酮可以促進這一過程，系膜細胞自噬激活可以抑制這種轉化。還有動物實驗證實，醛固酮可以造成腎臟細胞的衰老，而自噬正是體內細胞對抗衰老的主要生理過程。綜合上述的研究進展，醛固酮可能通過某些途徑對系膜細胞的自噬產生負性調控，而這種自噬活性的變化可能參與了系膜細胞的損傷應激及CKD的進展過程。 通過本研究，我們想進一步明確醛固酮對系膜細胞自噬的影響，初步的探討醛固酮對自噬的調控作用產生的生理及病理意義，積極探索可能參與其中的信號通路及分子機制。細胞的自噬往往與線粒體功能的變化及細胞內ROS的水平具有很大的關聯(lián)，而血管緊張素II及醛固酮都參與了系膜細胞線粒體的損傷及ROS的激活過程，我們也試圖進一步研究二者與系膜細胞線粒體損傷的關系。 實驗方法 1．使用CCK8方法及流式細胞術測定醛固酮及血管緊張素對于系膜細胞增殖的影響。 2．通過western blot方法檢測在饑餓、醛固酮、血管緊張素、表皮生長因子等不同干預的條件下系膜細胞自噬相關的蛋白標志物LC3、P62/SQSTM1的表達水平變化。檢測信號通路蛋白EGFR、ERK、GAB1、PARP及剪切體PARP、AMPK及Beclin-1等蛋白表達水平的變化。 3.光鏡下觀察H2O2刺激系膜細胞在有無醛固酮長期干預下的凋亡變化。 4．通過向系膜細胞轉入GFP-LC3的真核表達質粒，用激光共聚焦顯微鏡觀察不同的刺激影響下系膜細胞熒光自噬點數(shù)量的變化并進行統(tǒng)計學分析。 5.通過電子顯微鏡觀察不同的干預條件下系膜細胞自噬泡數(shù)量的變化，并進行統(tǒng)計學分析。 6.使用Mito-TrackerGreen探針（Invitrogen公司）檢測不同的干預條件下系膜細胞線粒體的數(shù)量變化。使用Mito-ID細胞外液酸度檢測試劑盒檢測不同干預條件下細胞外液酸度變化情況，顯示線粒體損傷后細胞糖酵解水平的變化。 7．免疫共沉淀的方法檢測醛固酮干預對于Beclin-1-BCL2復合體的影響。 8. Real-timePCR方法檢測了自噬相關基因及線粒體相關的基因在醛固酮及血管緊張素干預下的表達情況。 結果 1.在體外培養(yǎng)的腎小球系膜細胞系HMCL及RMC中，，高于正常濃度的醛固酮與血管緊張素II不能使兩種細胞系的增殖水平出現(xiàn)明顯的變化。流式細胞儀檢測干預后G2/M期細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的系膜細胞自噬活性的升高，且這種抑制表現(xiàn)為劑量依賴性。western blot檢測發(fā)現(xiàn)醛固酮干預后EBSS培養(yǎng)的系膜細胞LC3II表達降低及P62/SQSTM1表達升高。共聚焦顯微鏡觀察發(fā)現(xiàn)饑餓誘導的系膜細胞HMCL自噬點數(shù)量增加，而醛固酮可以抑制這種效應。電鏡下自噬泡的的計數(shù)觀察同樣證實醛固酮可以抑制饑餓誘導的系膜細胞自噬活化。 3.通過western blot、熒光顯微鏡觀察自噬點的形成以及電鏡結果均可證實雷帕霉素可以激活腎小球系膜細胞的自噬，醛固酮不能夠有效抑制這種自噬的激活效應。 4.在醛固酮導致的長期的自噬抑制狀態(tài)下，系膜細胞在面臨氧化應激時更容易發(fā)生凋亡。表現(xiàn)為在相同的時間點，凋亡細胞的比率明顯增加，western blot檢測發(fā)現(xiàn)PARP蛋白的剪切體的表達上升。 5.醛固酮、血管緊張素II能夠在5、15、60分鐘內輕度激活EGFR，促使其磷酸化的水平提高，但這種激活作用遠遠弱于EGF的直接刺激，三者激活EGFR的效應按強度大小排列為EGFAngIIAld。但EGF刺激EGFR磷酸化的強度隨時間很快衰減，而血管緊張素II與醛固酮對EGFR磷酸化則在1小時內緩慢增強，至48小時仍有持續(xù)的EGFR磷酸化及ERK的磷酸化。提示細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)，且同時具有時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位點的磷酸化，進而增加了其與BCL2的結合，導致Beclin-1與VPS34復合體結合減少，而抑制自噬。 7.與對照組及血清饑餓組相比，醛固酮干預24小時對于系膜細胞線粒體的數(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備功能，削弱系膜細胞線粒體對于應激的反應性，但血管緊張素II對其影響更大。 8.醛固酮及血管緊張素II均可以造成部分自噬相關基因的表達發(fā)生變化，其中血管緊張素II多造成這些基因表達的下調，而醛固酮則造成這些基因的表達上調。醛固酮對于部分線粒體相關的基因的表達影響較輕，而血管緊張素II的影響較明顯。特別是血管緊張素II對線粒體相關基因的影響程度大于醛固酮。結論 1.本實驗第一次發(fā)現(xiàn)了醛固酮對于系膜細胞饑餓誘導的自噬激活的抑制效應。這種抑制效應參與了醛固酮在氧化應激中對系膜細胞的協(xié)同損傷。 2.醛固酮抑制自噬效應的靶點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.

【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R692

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相關期刊論文 前1條

1 賴凌云,顧勇,陳靖,郁勝強,馬驥,楊海春,林善錟;大鼠系膜細胞醛固酮的合成及其對細胞外基質生成的影響[J];中華醫(yī)學雜志;2003年21期

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