發(fā)布時(shí)間：2018-09-19 13:40

【摘要】：背景：甲硫氨酸亞砜還原酶A(MsrA)是細(xì)胞內(nèi)特異還原S型甲硫氨酸亞砜(MetSO)的還原酶類(lèi),是細(xì)胞內(nèi)重要的抗氧化屏障。動(dòng)脈粥樣硬化所致的心腦血管疾病目前是世界致死、致殘率最高的一類(lèi)疾病,動(dòng)脈粥樣硬化是公認(rèn)的慢性炎癥性疾病,氧化應(yīng)激和血脂紊亂也是其主要的誘因。因此,抗氧化抗炎及降脂成為防治動(dòng)脈粥樣硬化的主要策略。細(xì)胞內(nèi)抗氧化酶MsrA與動(dòng)脈粥樣硬化發(fā)生發(fā)展的相關(guān)性并不十分明確,由于該蛋白酶必須在細(xì)胞內(nèi)依賴(lài)硫氧還蛋白系統(tǒng)恢復(fù)其活性,而作為生物大分子又難以直接穿過(guò)細(xì)胞膜進(jìn)入細(xì)胞內(nèi)發(fā)揮作用,目前利用外源性MsrA研究對(duì)動(dòng)脈粥樣硬化等氧化應(yīng)激所致疾病的作用及相關(guān)機(jī)制尚無(wú)報(bào)道。近年有報(bào)道,細(xì)胞穿膜肽PEP-1作為一種新型、有效、安全的工具肽,介導(dǎo)超氧化物歧化酶(SOD)、過(guò)氧化物酶(CAT)、對(duì)氧磷酯酶1(PON1)等抗氧化蛋白成功穿入細(xì)胞并發(fā)揮有效的抗氧化功能。本文擬構(gòu)建穿膜活性肽PEP-1與人源MsrA(hMsrA)的重組PEP-1-hMsrA融合蛋白,以巨噬細(xì)胞及載脂蛋白E基因敲除(apoE-/-)小鼠模型為研究對(duì)象,旨在探究PEP-1-hMsrA蛋白在動(dòng)脈粥樣硬化發(fā)生發(fā)展中的調(diào)節(jié)作用及可能的相關(guān)機(jī)制。方法：1.構(gòu)建含有hMsrA或PEP-1-hMsrA的重組原核表達(dá)載體pET28a/hMsrA及 pET28a/PEP-1-hMsrA,分別轉(zhuǎn)化至BL21DE3大腸桿菌、經(jīng)IPTG誘導(dǎo)表達(dá)及Ni-NTA親和層析獲得純品hMsrA及PEP-1-hMsrA蛋白。圓二色光譜法鑒定兩種蛋白的二級(jí)結(jié)構(gòu),并以DMSO為底物測(cè)定MsrA蛋白的還原酶活性。2.細(xì)胞實(shí)驗(yàn)：以不同濃度(0-18μM)的hMsrA或PEP-1-hMsrA蛋白孵育Hela細(xì)胞72h,MTT法測(cè)細(xì)胞存活率；以適宜的濃度及時(shí)間孵育小鼠巨噬細(xì)胞及人HepG2細(xì)胞,Western Blot及免疫熒光方法檢測(cè)PEP-1-hMsrA蛋白的穿膜效率；1 μ.M hMsrA或PEP-1-hMsrA蛋白分別孵育細(xì)胞1h后1mM過(guò)氧化氫(H202)刺激小鼠巨噬細(xì)胞,觀(guān)察PEP-1-hMsrA對(duì)氧化應(yīng)激下巨噬細(xì)胞內(nèi)ROS水平及細(xì)胞壞死的影響：熒光探針DCFH-DA檢測(cè)細(xì)胞內(nèi)活性氧簇(ROS)水平；AnnexinV-FITC/PI雙染細(xì)胞凋亡試劑盒檢測(cè)細(xì)胞凋亡壞死率：25ng/ml脂多糖(LPS)與1μM hMsrA或PEP-1-hMsrA蛋白共孵育小鼠巨噬細(xì)胞,RT-PCR檢測(cè)細(xì)胞內(nèi)炎癥相關(guān)基因IL-1β、TNFα、IL-10的轉(zhuǎn)錄水平。3.動(dòng)物實(shí)驗(yàn)：選擇21周齡雄性apoE-/-小鼠作為實(shí)驗(yàn)對(duì)象,經(jīng)腹腔連續(xù)注射5.5nmol hMsrA或PEP-1-hMsrA蛋白,設(shè)空白組注射等體積磷酸鹽緩沖液(100 mMPBS,pH7.4),每36h一次。實(shí)驗(yàn)中給予小鼠高脂飲食加速動(dòng)脈粥樣硬化進(jìn)程,于第12周最后一次注射蛋白2h后小鼠行安樂(lè)死。采取禁食12h后小鼠全血分離血漿,采用小鼠主動(dòng)脈竇冰凍切片、主動(dòng)脈弓及主動(dòng)脈、油紅0染色進(jìn)行斑塊橫切面及en face剖面分析主動(dòng)脈粥樣硬化斑塊面積；酶法測(cè)定血清總膽固醇(TC)和甘油三酯(TG)濃度；利用免疫組化及TUNEL染色檢測(cè)主動(dòng)脈斑塊中巨噬細(xì)胞數(shù)量及細(xì)胞凋亡比例；ELISA法測(cè)定血清炎癥因子MCP-1蛋白水平,RT-PCR檢測(cè)肝臟組織中炎癥因子TNFα和IL-6的mRNA水平；檢測(cè)血清PON1和SOD活性,RT-PCR及Western Blot同時(shí)檢測(cè)肝臟組織中PONI的表達(dá)水平。結(jié)果：1.成功構(gòu)建重組原核表達(dá)載體pET28a/hMsrA和pET28a/PEP-1-hMsrA,DNA測(cè)序證實(shí)堿基序列正確；S DS-PAGE鑒定經(jīng)親和層析獲得的hMsrA和PEP-1-hMsrA蛋白純度均高于95%,得率約為20 mg/L菌液(菌體重量約為5g)；圓二色光譜法鑒定,hMsrA和PEP-1-hMsrA蛋白的α螺旋結(jié)構(gòu)無(wú)明顯差異(18.1%vs.17.1%)；體外對(duì)比MsrA的還原酶活性,結(jié)果顯示hMsrA和PEP-1-hMsrA的酶活性無(wú)明顯差異。2.細(xì)胞實(shí)驗(yàn)中發(fā)現(xiàn)即使高濃度(18pM)的hMsrA或PEP-1-hMsrA蛋白長(zhǎng)時(shí)間(72h)孵育Hela,對(duì)細(xì)胞活力沒(méi)有影響：以不同濃度蛋白孵育巨噬細(xì)胞或人肝細(xì)胞,蛋白免疫印跡及激光共聚焦均證實(shí)PEP-1-hMsrA蛋白具備有效穿膜效應(yīng)且表現(xiàn)一定的濃度依賴(lài)性,在細(xì)胞中可持續(xù)存在達(dá)12h。與hMsrA組相比,PEP-1-hMsrA蛋白可顯著降低H202誘發(fā)的胞內(nèi)ROS水平,并明顯降低H202引起的細(xì)胞壞死率及凋亡率；LPS刺激炎癥相關(guān)基因IL-1β、TNFα、IL-10 mRNA的表達(dá),PEP-1-hMsrA蛋白可顯著降低胞內(nèi)致炎因子I]L-1β、TNFα的mRNA水平,但可以明顯提高抗炎因子IL-10的mRNA水平。3.動(dòng)物實(shí)驗(yàn)中,與注射PBS的對(duì)照組比較,hMsrA和PEP-1-hMsrA蛋白注射對(duì)小鼠體重、脾重、血漿TC、TG、IgG水平并無(wú)明顯影響；但P EP-1-hMsrA組可顯著提高血中抗氧化蛋白PON1、SOD的活性,并降低血清中炎癥因子MCP-1的蛋白水平；與PBS對(duì)照組及hMsrA組比較,給予PEP-1-hMsrA蛋白可明顯降低高脂喂養(yǎng)下小鼠主動(dòng)脈竇部粥樣斑塊面積,同時(shí)顯著降低主動(dòng)脈弓和主動(dòng)脈的粥樣斑塊面積比例,并使主動(dòng)脈竇部斑塊中巨噬細(xì)胞數(shù)量減少,并且細(xì)胞的凋亡比率也明顯降低；同時(shí)發(fā)現(xiàn),PEP-1-hMsrA蛋白間接地提高了小鼠肝臟中PON1的mRNA和蛋白表達(dá)水平；肝臟中炎癥因子TNFa及IL-6的mRNA水平也下降。結(jié)論：成功構(gòu)建并獲得具有穿膜效應(yīng)的PEP-1-hMs rA蛋白,證實(shí)其可明顯提高巨噬細(xì)胞的抗氧化、抗炎能力,并顯著減緩高脂飲食下apoE-/-小鼠動(dòng)脈粥樣硬化斑塊的發(fā)展進(jìn)程,其機(jī)制可能與PEP-1-hMsrA進(jìn)入血管、肝組織細(xì)胞,改善局部及血循環(huán)系統(tǒng)的氧化、炎癥狀態(tài)有關(guān)。本研究為動(dòng)脈粥樣硬化的抗氧化防治提供了新的策略和依據(jù)。
[Abstract]:BACKGROUND: Methionine sulfoxide reductase A (MsrA) is a reductase that specifically reduces S-type methionine sulfoxide (MetSO) in cells and an important antioxidant barrier in cells. Oxidative stress and dyslipidemia are also major contributors to atherosclerosis. Therefore, antioxidant, anti-inflammatory and lipid-lowering are the main strategies for preventing and treating atherosclerosis. However, as a biological macromolecule, it is difficult to directly penetrate the cell membrane into the cell to play a role. Exogenous MsrA has not been reported to study the role of oxidative stress induced diseases such as atherosclerosis and related mechanisms. Antioxidant proteins such as dismutase (SOD), peroxidase (CAT) and paraoxonase-1 (PON1) successfully penetrate into cells and play an effective antioxidant role. In this study, the recombinant PEP-1-hMsrA fusion protein of PEP-1 and human MsrA (hMsrA) was constructed. The macrophages and apoE-knockout mice were used as the research objects. Methods: 1. Recombinant prokaryotic expression vectors containing hMsrA or PEP-1-hMsrA, pET28a/hMsrA and pET28a/PEP-1-hMsrA, were constructed and transformed into E. coli BL21DE3 by IPTG induction and Ni-NTA affinity chromatography. The secondary structures of the two proteins were identified by circular dichroism spectroscopy, and the reductase activity of MsrA protein was measured by DMSO as substrate. 2. Cell experiments: Hela cells were incubated with different concentrations of hMsrA or PEP-1-hMsrA protein for 72 hours, and the cell viability was measured by MTT assay. The penetration efficiency of PEP-1-hMsrA protein was detected by Western Blot and immunofluorescence assay in human HepG2 cells, and the effects of PEP-1-hMsrA protein on ROS level and cell necrosis in macrophages under oxidative stress were observed by DCFH-DA with fluorescent probe. Cellular reactive oxygen species (ROS) level was measured; Annexin V-FITC/PI double staining apoptosis kit was used to detect apoptosis and necrosis rate: 25ng/ml lipopolysaccharide (LPS) was co-incubated with 1 mu mhMsrA or PEP-1-hMsrA protein in mouse macrophages, and RT-PCR was used to detect the transcription level of inflammation-related genes IL-1beta, TNF-a and IL-10.3. Animal experiment: 21 weeks old. Male apoE-/-mice were injected with 5.5 nmol hMsrA or PEP-1-hMsrA protein through abdominal cavity. The blank group was injected with equal volume phosphate buffer (100 mMPBS, pH 7.4) every 36 hours. The mice were given a high fat diet to accelerate the atherosclerosis process. The mice were euthanized 2 hours after the last injection of protein at the 12th week. After fasting for 12 hours, the plasma was isolated from the whole blood of mice. The aortic sinus, aortic arch and aorta were stained with oil red 0, and the plaque area was analyzed by transverse section and en face section. The serum total cholesterol (TC) and triglyceride (TG) levels were measured by enzyme method, and the aorta was detected by immunohistochemistry and TUNEL staining. The number of macrophages and the proportion of apoptosis in the plaque were measured by ELISA. The levels of serum inflammatory factors MCP-1 protein were measured by ELISA. The mRNA levels of inflammatory factors TNFalpha and IL-6 in liver tissue were detected by RT-PCR. The activities of serum PON1 and SOD were detected. The expression of PONI in liver tissue was detected by RT-PCR and Western Blot. The prokaryotic expression vectors pET28a/hMsrA and pET28a/PEP-1-hMsrA were confirmed by DNA sequencing. The purity of hMsrA and PEP-1-hMsrA proteins obtained by affinity chromatography identified by S DS-PAGE was higher than 95%, and the yield was about 20 mg/L bacterial fluid (body weight was about 5 g); circular dichroism spectroscopy showed that the alpha helix structure of hMsrA and PEP-1-hMsrA proteins was not obvious. The activity of MsrA and PEP-1-hMsrA was not significantly different in vitro. 2. Cell experiments showed that even high concentration (18pM) of hMsrA or PEP-1-hMsrA protein incubated Hela for a long time (72h) had no effect on cell viability: macrophages or human hepatocytes were incubated with different concentrations of proteins. PEP-1-hMsrA protein could effectively penetrate the membrane and exhibit a certain concentration-dependent effect. It could persist for 12 hours. Compared with hMsrA group, PEP-1-hMsrA protein could significantly reduce the intracellular ROS level induced by H202 and the cell necrosis rate and apoptosis rate induced by H202. The expression of IL-1beta, TNFalpha, IL-10 mRNA and PEP-1-hMsrA protein could significantly decrease the mRNA levels of intracellular inflammatory factors I]L-1beta and TNFalpha, but could significantly increase the mRNA levels of anti-inflammatory factors IL-10. 3. In animal experiments, compared with the control group injected with PBS, hMsrA and PEP-1-hMsrA protein injected mice weight, spleen weight, plasma TC, TG, I. There was no significant effect on the level of gG, but PEP-1-hMsrA significantly increased the activity of antioxidant protein PON1 and SOD, and decreased the level of inflammatory factor MCP-1 in serum. Compared with PBS control group and hMsrA group, PEP-1-hMsrA protein significantly decreased the area of atherosclerotic plaque in aortic sinus of mice fed with high-fat diet, and also significantly decreased the area of atherosclerotic plaque. The proportion of atherosclerotic plaque area in aortic arch and aorta decreased the number of macrophages in aortic sinus plaque and the rate of apoptosis of the cells. It was also found that PEP-1-hMsrA protein indirectly increased the expression of PON1 mRNA and protein in mice liver, and the levels of inflammatory factors TNFa and IL-6 mRNA in liver were also increased. CONCLUSION: PEP-1-hMs rA protein with membrane-penetrating effect was successfully constructed and obtained, which proved that PEP-1-hMs rA protein could significantly improve the antioxidant and anti-inflammatory capacity of macrophages, and significantly slow down the development of atherosclerotic plaques in apoE-/-mice fed with high-fat diet. The mechanism may be related to the entry of PEP-1-hMs rA into blood vessels, hepatocytes, and the improvement of local and blood levels. This study provides a new strategy and basis for the antioxidant prevention and treatment of atherosclerosis.
【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(lèi)號(hào)】：R543.5

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