發(fā)布時(shí)間：2018-06-25 06:58

  本文選題：MsrA + 甲硫氨酸亞砜��； 參考：《武漢大學(xué)》2015年博士論文

【摘要】：研究背景脂質(zhì)代謝紊亂和炎癥是動(dòng)脈粥樣硬化(atherosclerosis, As)形成的公認(rèn)危險(xiǎn)因素。肝臟作為機(jī)體脂質(zhì)代謝和炎癥控制的重要器官,與As的發(fā)生發(fā)展密切相關(guān)。調(diào)節(jié)肝臟脂質(zhì)代謝及炎癥是防治As的有效策略。最新研究發(fā)現(xiàn)甲硫氨酸殘基氧化修飾(甲硫氨酸亞砜)可通過影響某些特定蛋白質(zhì)的結(jié)構(gòu)與功能,參與多種氧化相關(guān)疾病的發(fā)生與發(fā)展。而MsrA作為機(jī)體內(nèi)一道特殊的蛋白抗氧化防護(hù)屏障,可特異性還原S型甲硫氨酸亞砜(Methionine S-sulfoxide, MetSO),在調(diào)節(jié)蛋白功能及細(xì)胞氧還狀態(tài)平衡的過程中扮演重要的角色。因此,本研究擬將MsrA作為一個(gè)調(diào)控靶點(diǎn),干預(yù)肝臟氧還狀態(tài),觀察肝臟hMsrA高表達(dá)對(duì)As模型鼠炎癥、脂質(zhì)代謝及As斑塊的影響并探究其相關(guān)機(jī)制。方法體外研究：采用脂質(zhì)體法分別將構(gòu)建的慢病毒表達(dá)載體質(zhì)粒pWPI-hMsrA-GFP及對(duì)照質(zhì)粒pWPI-GFP瞬時(shí)轉(zhuǎn)染傳代培養(yǎng)的人肝癌HepG2細(xì)胞系,熒光顯微鏡觀察細(xì)胞報(bào)告基因GFP表達(dá)狀態(tài),蛋白免疫印跡檢測(cè)MsrA表達(dá)水平。利用透膜熒光探針二氫溴乙啶(氧化轉(zhuǎn)化為溴化乙啶)熒光顯微鏡下檢測(cè)細(xì)胞紅色及綠色熒光,ImageJ軟件計(jì)算轉(zhuǎn)染細(xì)胞的平均熒光強(qiáng)度分析胞內(nèi)活性氧簇(ROS)的水平;實(shí)時(shí)定量PCR (Q-PCR)及蛋白免疫印跡檢測(cè)HepG2細(xì)胞炎癥和脂質(zhì)代謝相關(guān)基因表達(dá)的水平,觀察hMsrA高表達(dá)對(duì)HepG2細(xì)胞炎癥及脂代謝的影響。體內(nèi)研究：將慢病毒表達(dá)載體pWPI-GFP/pWPI-hMsrA-GFP和包裝質(zhì)粒PCMV/PD2G,通過磷酸鈣法共轉(zhuǎn)染293T細(xì)胞,包裝形成慢病毒顆粒(Lv-GFP/Lv-MsrA-GFP),收集、濃縮獲得高滴度慢病毒顆粒濃縮液。先后采用載脂蛋白E基因敲除(apoE-/-)小鼠或清道夫受體BI基因敲除(SR-BI-/-)小鼠作為As模型鼠,各自隨機(jī)分兩組,每組8只,分別經(jīng)眼球后靜脈叢注射pWPI-GFP慢病毒(Lv-GFP對(duì)照組)和含hMsrA的重組慢病毒(Lv-MsrA-GFP組)；慢病毒顆粒輸注兩周后,小鼠采用AIN-76A西方膳食高脂喂養(yǎng)12周,加速As進(jìn)程。實(shí)驗(yàn)小鼠自病毒注射兩周后每間隔3-4周采血,酶法監(jiān)測(cè)血漿總膽固醇(TC)、甘油三酯(TG)、游離膽固醇(FC)、高密度脂蛋白膽固醇(HDL-C)等血脂水平；快速蛋白液相色譜(fast protein liquid chromatography, FPLC)分析血漿脂蛋白膽固醇分布；血漿超氧化物歧化酶(SOD)采用試劑盒檢測(cè)；血漿對(duì)氧磷酯酶(PON1)活性利用對(duì)氧磷為底物,采用連續(xù)動(dòng)態(tài)法監(jiān)測(cè)；異丙醇法抽提糞便脂質(zhì)并檢測(cè)糞膽固醇含量。病毒注射14周后小鼠安樂死,從主動(dòng)脈根部連續(xù)冰凍切片及主動(dòng)脈胸腹段en face剖面進(jìn)行油紅O染色分析斑塊面積。采取小鼠肝臟組織行組織化學(xué)檢測(cè)肝臟MsrA表達(dá)水平；酶法分析肝臟脂質(zhì)含量；實(shí)時(shí)熒光定量PCR檢測(cè)小鼠肝臟炎癥及脂質(zhì)代謝相關(guān)基因的mRNA水平；蛋白免疫印跡檢測(cè)肝臟脂質(zhì)代謝相關(guān)基因和血漿中載脂蛋白AI(apoAI)、PON1及血清淀粉樣蛋白A(SAA)的蛋白水平。結(jié)果1. hMsrA高表達(dá)可降低HepG2細(xì)胞ROS水平,抑制炎癥因子腫瘤壞死因子α(TNFα)和白細(xì)胞介素-6(IL-6)的產(chǎn)生,也可顯著上調(diào)膽固醇代謝相關(guān)基因肝X受體α(LXRα)、ATP結(jié)合盒轉(zhuǎn)運(yùn)蛋白Al(ABCA1)及SR-BI的基因表達(dá),但對(duì)低密度脂蛋白受體(LDLR)的基因表達(dá)未產(chǎn)生明顯影響。結(jié)果表明高水平hMsrA可通過發(fā)揮抗氧化作用,降低ROS水平,影響HepG2細(xì)胞炎癥及脂代謝相關(guān)基因表達(dá)。2.GFP熒光檢測(cè)及MsrA的免疫組化均顯示,慢病毒注射可使小鼠肝臟高表達(dá)hMsrA,實(shí)驗(yàn)中小鼠的體重、脾重及血漿谷丙轉(zhuǎn)氨酶(ALT)活性無明顯變化,說明肝臟高表達(dá)hMsrA并未對(duì)小鼠免疫及肝功能產(chǎn)生明顯不良影響。與Lv-GFP小鼠相比,Lv-MsrA-GFP apoE-/-小鼠肝臟MsrA高表達(dá)3.4倍;而Lv-MsrA-GFP組SR-BI-/-小鼠肝臟MsrA水平是對(duì)照組SR-BIA鼠的2.38倍,即肝臟hMsrA高表達(dá)實(shí)驗(yàn)動(dòng)物模型構(gòu)建成功。3.肝臟hMsrA高表達(dá)對(duì)高脂喂養(yǎng)apoE-/-小鼠的影響：1)與Lv-GFP組相比,Lv-MsrA-GFP組小鼠肝臟炎癥因子TNFa/IL-6的mRNA水平明顯下調(diào),血漿抗氧化酶SOD、PON1的活性顯著提高,血漿PON1、apoAI的蛋白含量也明顯增加,而血漿急性應(yīng)激蛋白因子SAA水平顯著下降；2)肝臟hMsrA高表達(dá)可明顯降低TC、TG,FPLC分析脂蛋白分布可見血脂水平主要是血漿VLDL/LDL水平的降低,HDL-C并未見顯著變化；3)肝臟hMsrA高表達(dá)可通過上調(diào)肝臟膽固醇代謝相關(guān)基因SR-BI、apoAI、LXRa、ABCA1/G8及膽固醇7/27α-羥化酶(CYP7A1/27A1)的表達(dá),并協(xié)同調(diào)節(jié)肝臟膽固醇酯水解酶(CEH)和乙酰輔酶A乙酰轉(zhuǎn)移酶(ACAT)的mRNA和/或蛋白水平,顯著增強(qiáng)apoE-/-小鼠肝臟對(duì)膽固醇的選擇性攝取、轉(zhuǎn)化及膽道排泄,提高糞便膽固醇的含量(增加24.6%),減少肝臟脂質(zhì)沉積；4)肝臟hMsrA高表達(dá)顯著下調(diào)脂肪合成相關(guān)酶：乙酰輔酶A羧化酶a (ACCa)/脂肪酸合成酶(FASN)的mRNA水平,抑制TG合成,降低肝臟及血漿TG含量；5)肝臟hMsrA高表達(dá)可顯著減緩高脂喂養(yǎng)的apoE-/-小鼠動(dòng)脈粥樣硬化斑塊的進(jìn)程：與Lv-GFP組相比,Lv-MsrA-GFP組實(shí)驗(yàn)小鼠主動(dòng)脈根部橫截面切片的平均斑塊面積明顯減少(0.50±0.06 vs 0.66±0.06 mm2; P0.01);胸腹主動(dòng)脈en face剖面分析結(jié)果顯示,Lv-MsrA-GFP組小鼠主動(dòng)脈弓及主動(dòng)脈斑塊面積均明顯減小(P0.01,P0.05)。上述結(jié)果提示：肝臟hMsrA高表達(dá)可顯著改善apoE-/-小鼠肝臟及循環(huán)中的氧還狀態(tài),抑制炎癥,并促進(jìn)肝臟膽固醇攝取、轉(zhuǎn)化及膽道排泄,抑制TG合成,從而改善脂質(zhì)代謝,降低肝臟脂質(zhì)沉積,阻抑As斑塊進(jìn)程。4.肝臟hMsrA高表達(dá)對(duì)高脂喂養(yǎng)SR-BI-/-小鼠的影響：1)與Lv-GFP組相比,Lv-MsrA-GFP組小鼠肝臟炎癥因子TNFa/IL-6的mRNA水平明顯下調(diào),血漿急性蛋白因子SAA水平明顯降低,而血漿抗氧化酶PON1的活性顯著提高。2)SR-BI-/-小鼠肝臟hMsrA高表達(dá)可明顯降低血漿TG、FC,提升血漿HDL-C水平,但對(duì)血漿TC影響不明顯；3) SR-BI-/-鼠肝臟hMsrA高表達(dá)也可通過下調(diào)ACCa/FASN的表達(dá),抑制TG合成；4) SR-BI-/-鼠肝臟hMsrA高表達(dá)不再上調(diào)肝臟膽固醇代謝相關(guān)基因apoAI、LXRa-ABCA1/G8及CYP7A1/27A1的表達(dá),不改變小鼠糞便/肝臟膽固醇含量；但肝臟hMsrA高表達(dá)可通過上調(diào)肝臟ACAT、 LXRa、ABCA1及血漿apoAI蛋白水平,與降低高脂喂養(yǎng)SR-BI-/-鼠肝臟和血漿FC水平有關(guān)。以上結(jié)果證實(shí)：肝臟hMsrA高水平改善SR-BI小鼠氧還狀態(tài),抑制炎癥,并抑制TG合成,降低肝臟/血漿FC比例,但不改變血漿/肝臟/糞便TC水平,原因可能與小鼠SR-BI缺陷無法調(diào)節(jié)肝臟膽固醇的選擇攝取有關(guān)。結(jié)論MsrA作為胞內(nèi)一種特殊抗氧化酶,其肝臟高表達(dá)可顯著降低小鼠肝臟組織局部和機(jī)體循環(huán)系統(tǒng)的氧化狀態(tài),發(fā)揮抗炎和調(diào)節(jié)脂質(zhì)代謝的作用,從而降低小鼠肝臟脂質(zhì)沉積,及阻抑動(dòng)脈粥樣硬化斑塊的進(jìn)程。其調(diào)脂作用主要涉及：促進(jìn)SRBI介導(dǎo)的肝臟膽固醇攝取、膽固醇轉(zhuǎn)化及膽固醇膽道排泄而降肝臟/血漿膽固醇水平；降低TG合成,一定條件下協(xié)調(diào)膽固醇的酯化及膽道排泄過程�？傊�,肝臟MsrA可有效抗氧化抗炎、改善脂質(zhì)代謝,有望成為防治動(dòng)脈粥樣硬化相關(guān)心血管疾病的重要靶向蛋白。
[Abstract]:Background lipid metabolism disorder and inflammation are recognized as a risk factor for the formation of atherosclerosis (As). The liver is an important organ for lipid metabolism and inflammation control, which is closely related to the development of As. Regulation of liver lipid metabolism and inflammation is an effective strategy for the prevention and treatment of As. The latest research has found methionine residues Oxidative modification (methionine sulfoxide) can participate in the occurrence and development of a variety of oxidative related diseases by affecting the structure and function of certain specific proteins, and MsrA, as a special antioxidant protective barrier in the body, can specifically reduce S type methionine sulfoxide (Methionine S-sulfoxide, MetSO), and regulate protein function and the function of protein. Cell oxygen also plays an important role in the process of balance. Therefore, this study intends to use MsrA as a regulatory target to intervene in the state of liver oxygenation, to observe the effect of liver hMsrA high expression on inflammation, lipid metabolism and As plaque in As model rats and to explore its related mechanisms. The virus expression vector plasmid pWPI-hMsrA-GFP and the control plasmid pWPI-GFP were transiently transfected to the human hepatoma HepG2 cell line. The expression of the cell reporter gene GFP was observed by the fluorescence microscope and the expression level of MsrA was detected by the protein immunoblotting. The fluorescence microscopy of the transmembrane fluorescence probe two hydrobromide (oxidation conversion to ethidium bromide) was used for the fluorescence microscopy. Cell red and green fluorescence were measured. ImageJ software calculated the average fluorescence intensity of transfected cells to analyze the level of intracellular reactive oxygen species (ROS); real-time quantitative PCR (Q-PCR) and protein immunoblotting were used to detect the expression level of HepG2 cell inflammation and lipid metabolism related genes. The effect of hMsrA high expression on the inflammation and lipid metabolism of HepG2 cells was observed. Study: the Lentivirus Expression Vector pWPI-GFP/pWPI-hMsrA-GFP and the package plasmid PCMV/PD2G were transfected into 293T cells by calcium phosphate method and packaged to form lentivirus particles (Lv-GFP/Lv-MsrA-GFP) and collected and concentrated to obtain high titer lentivirus particle concentrate. It was successively adopted the fat egg white E gene knockout (apoE-/-) mouse or the scavenger receptor BI gene successively. SR-BI-/- mice were randomly divided into two groups, each of which was randomly divided into two groups, 8 in each group. The mice were injected with pWPI-GFP lentivirus (Lv-GFP control group) and hMsrA containing recombinant lentivirus (group Lv-MsrA-GFP), respectively. After two weeks of lentivirus particles infusion, the mice were fed with AIN-76A Western diet high fat for 12 weeks and accelerated As process. Experimental mice were taken from mice. Blood samples were collected every 3-4 weeks after two weeks of injection. Plasma total cholesterol (TC), triglyceride (TG), free cholesterol (FC), high density lipoprotein cholesterol (HDL-C) and other lipid levels were monitored by enzyme method. Plasma lipoprotein cholesterol distribution was analyzed by rapid protein liquid chromatography (fast protein liquid chromatography, FPLC); plasma superoxide dismutase (SOD) (SOD) use the kit test; plasma oxyphosphatase (PON1) activity using oxyphosphoric acid as the substrate, using continuous dynamic monitoring, isopropyl alcohol extraction of fecal lipid and detection of fecal cholesterol content. After 14 weeks of virus injection, mice were euthanized, the continuous frozen section of the aorta and the en face section of the thoracic and abdominal segment of the aorta were stained with oil red O staining. Color analysis of plaque area. The liver MsrA expression level was detected by mouse liver tissue; liver lipid content was analyzed by enzyme method; real-time fluorescence quantitative PCR was used to detect the mRNA level of liver inflammation and lipid metabolism related genes in mice; liver lipid metabolism related genes and plasma apolipoprotein AI (apoAI) were detected by Western blot. The protein level of PON1 and serum amyloid A (SAA). Results the high expression of 1. hMsrA can reduce the ROS level of HepG2 cells, inhibit the production of tumor necrosis factor alpha (TNF a) and interleukin -6 (IL-6), and also significantly up-regulate the cholesterol metabolism related gene liver X receptor alpha (LXR alpha), ATP binding box transporter and substrate. The expression has no obvious effect on the gene expression of low density lipoprotein receptor (LDLR). The results show that high level hMsrA can reduce the level of ROS by exerting antioxidant activity, affecting the expression of.2.GFP fluorescence in HepG2 cell inflammation and lipid metabolism related gene expression and immunohistochemistry of MsrA. Up to hMsrA, the weight of mice, spleen weight and plasma alanine transaminase (ALT) activity did not change significantly, indicating that the liver high expression hMsrA did not have a significant adverse effect on the immune and liver function of mice. Compared with the Lv-GFP mice, the MsrA in the liver of Lv-MsrA-GFP apoE-/- mice was 3.4 times higher than that in the Lv-MsrA-GFP group, and the MsrA level of the liver of Lv-MsrA-GFP group SR-BI-/- mice was In the control group, 2.38 times the SR-BIA rat, that is, the liver hMsrA high expression experimental animal model was constructed successfully. The effect of the high expression of.3. liver hMsrA on the high fat feeding apoE-/- mice: 1) the mRNA level of the liver inflammatory factor TNFa/IL-6 in the Lv-MsrA-GFP group was significantly lower than that in the Lv-GFP group, and the activity of the plasma antioxidant enzyme SOD, PON1 was significantly increased, and the plasma PON1, and the plasma PON1, were The protein content of poAI increased significantly, while the level of acute stress protein factor SAA in plasma decreased significantly; 2) high expression of hMsrA in the liver could significantly reduce TC, TG, FPLC analysis of lipoprotein distribution showed that blood lipid level was mainly the decrease of plasma VLDL/LDL level, and no significant change in HDL-C, and 3) liver hMsrA high expression could be raised by up regulation of liver cholesterol. The expression of metabolic related genes SR-BI, apoAI, LXRa, ABCA1/G8 and cholesterol 7/27 alpha hydroxylase (CYP7A1/27A1), and co regulated mRNA and / or protein levels of liver cholesterol ester hydrolase (CEH) and acetyl coenzyme A acetyltransferase (ACAT), significantly enhanced the selective uptake of cholesterol in the liver of apoE-/- mice, transformation and biliary excretion, and increased feces. The content of cholesterol (increased by 24.6%), reduced liver lipid deposition, 4) liver hMsrA high expression significantly down regulation of lipid synthesis related enzymes: acetyl coenzyme A carboxylase a (ACCa) / fatty acid synthase (FASN) mRNA level, inhibition of TG synthesis, reducing the liver and plasma TG content; 5) liver hMsrA high expression can significantly slow down high fat feeding apoE-/- small apoE-/- The process of atherosclerotic plaque in rats: compared with the Lv-GFP group, the average plaque area of the aortic root section of the Lv-MsrA-GFP group was significantly reduced (0.50 + 0.06 vs 0.66 + 0.06 mm2; P0.01). The results of the thoracic and abdominal aorta en face profile analysis showed that the area of aortic arch and aortic plaque in Lv-MsrA-GFP mice was obvious. Decrease (P0.01, P0.05). These results suggest that the high expression of hMsrA in the liver can significantly improve the state of oxygen in the liver and circulation of apoE-/- mice, inhibit inflammation, promote liver cholesterol uptake, transform and bile duct excretion, inhibit TG synthesis, thus improve lipid metabolism, reduce liver lipid deposition, and inhibit the high expression of hMsrA in the As plaque process of.4.. The effect on high fat feeding SR-BI-/- mice: 1) compared with the Lv-GFP group, the mRNA level of liver inflammatory factor TNFa/IL-6 in the Lv-MsrA-GFP group was obviously down, the plasma acute protein factor SAA level was significantly reduced, and the activity of the plasma antioxidant enzyme PON1 was significantly increased.2) and the high expression of hMsrA in the liver of the SR-BI-/- mice could obviously reduce the TG, FC, and ascension of the plasma. The plasma HDL-C level was not significantly affected by the plasma TC; 3) the high expression of hMsrA in the liver of SR-BI-/- rats could also reduce the expression of ACCa/FASN and inhibit the synthesis of TG; 4) the high expression of hMsrA in the liver of SR-BI-/- mice no longer up-regulated the expression of cholesterol metabolism related genes of liver, LXRa-ABCA1/G8 and CYP7A1 /27A1, and did not change the cholesterol and liver cholesterol of mice. The high expression of hMsrA in the liver can be related to the increase of liver ACAT, LXRa, ABCA1 and plasma apoAI protein levels, which are related to the reduction of FC levels in the liver and plasma of SR-BI-/- rats fed by high fat diet. The above results confirm that the high level of liver hMsrA improves the oxygen state of the SR-BI mice, inhibits the inflammation, inhibits the synthesis of TG, reduces the FC proportion of the liver / plasma, but does not change the liver / plasma FC ratio. The level of plasma / liver / fecal TC may be related to the failure to regulate the selective uptake of liver cholesterol by SR-BI deficiency in mice. Conclusion MsrA is a special antioxidant enzyme in the cell. The high expression of the liver can significantly reduce the oxidation state of the local and circulatory system of the liver tissue in mice, and play the role of anti-inflammatory and regulating lipid metabolism. The lipid deposition of the liver and the inhibition of atherosclerotic plaques are reduced. The lipid regulating effect is mainly involved in promoting SRBI mediated cholesterol uptake, cholesterol conversion and cholesterol excretion, reducing liver / plasma cholesterol levels, reducing TG synthesis, and coordinating cholesterol esterification and biliary excretion under certain conditions. In conclusion, liver MsrA is an effective target protein for the prevention and treatment of atherosclerotic cardiovascular diseases, which is effective in anti-oxidation, anti-inflammatory and improving lipid metabolism.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R543.5

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1 潘曙光;MsrA在胃癌組織中的表達(dá)及意義[D];中南大學(xué);2009年

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