本文選題：Clq/腫瘤壞死因子相關(guān)蛋白9 + 動(dòng)脈粥樣硬化　； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：1研究背景心腦血管疾病是目前世界范圍內(nèi)致殘、致死的首位原因,其病理基礎(chǔ)主要是動(dòng)脈粥樣硬化(atherosclerosis,AS)。大量研究已證實(shí)AS是一種主要累及大中血管壁的慢性炎癥和代謝性疾病。AS的病理生理過(guò)程十分復(fù)雜,目前其發(fā)病機(jī)制仍有待于進(jìn)一步闡明。自噬是真核生物在進(jìn)化過(guò)程中高度保守的依賴溶酶體降解細(xì)胞內(nèi)自身細(xì)胞器和蛋白質(zhì)的自我保護(hù)過(guò)程。隨著研究的進(jìn)展,目前認(rèn)為自噬在AS發(fā)生發(fā)展過(guò)程中扮演著重要的角色,多種AS危險(xiǎn)因素均可引起自噬功能紊亂。自噬在AS中可以參與調(diào)節(jié)氧化應(yīng)激、代謝、炎癥和凋亡等過(guò)程。適度水平的自噬對(duì)AS起保護(hù)性作用,因此調(diào)節(jié)自噬水平或許能成為治療動(dòng)脈粥樣硬化相關(guān)性心血管疾病的重要靶點(diǎn)。哺乳動(dòng)物雷帕霉素靶蛋白(the mammalian target of rapamycin,mTOR)是雷帕霉素在哺乳動(dòng)物細(xì)胞內(nèi)的靶分子,mTOR在細(xì)胞內(nèi)存在mTORC1和mTORC2兩種復(fù)合體,其中mTORC1參與調(diào)節(jié)多種細(xì)胞的生長(zhǎng)和自噬等過(guò)程。抑制mTORC1可以通過(guò)多種途徑發(fā)揮AS保護(hù)作用,如改善內(nèi)皮功能紊亂、抑制單核細(xì)胞粘附和聚集、減少斑塊內(nèi)泡沫細(xì)胞形成、降低巨噬細(xì)胞炎癥反應(yīng)以及延緩平滑肌細(xì)胞增殖和遷移等。腺苷酸活化蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)作為真核細(xì)胞的一個(gè)重要能量感受器,活化后可以負(fù)性調(diào)節(jié)mTORC1的活性,從而上調(diào)自噬水平。越來(lái)越多的證據(jù)顯示,活化AMPK或者抑制mTOR或?yàn)閯?dòng)脈粥樣硬化防治的新靶點(diǎn)。C1q/腫瘤壞死因子相關(guān)蛋白 9(C1q/TNF-relatedprotein9,CTRP9)是 CTRP超家族中的新型脂肪細(xì)胞因子,與脂聯(lián)素(adiponectin,APN)高度同源。研究顯示CTRP9在調(diào)節(jié)代謝、保護(hù)心肌、改善內(nèi)皮功能及抵抗糖尿病心血管損害等方面發(fā)揮重要作用。越來(lái)越多的研究展現(xiàn)出了 CTRP9對(duì)心血管系統(tǒng)的保護(hù)性作用。CTRP9可以通過(guò)調(diào)控平滑肌細(xì)胞增殖和遷移過(guò)程延緩血管損傷后病理性血管重塑。同時(shí),CTRP9可以通過(guò)抑制心肌細(xì)胞凋亡和炎癥反應(yīng)降低心肌損傷。此外,血清CTRP9水平可作為一個(gè)獨(dú)立于APN的預(yù)測(cè)因素評(píng)估心血管事件的發(fā)生。我們前期研究顯示,與健康對(duì)照人群相比,急性冠脈綜合癥(acute coronary syndrome,ACS)患者血清中CTRP9和APN水平均降低,并且CTRP9和APN的血清水平與ACS病變嚴(yán)重程度呈負(fù)相關(guān)。我們進(jìn)一步發(fā)現(xiàn)在AS易損斑塊模型小鼠中,CTRP9可增加AS易損斑塊的穩(wěn)定性。近期研究顯示,在人原代培養(yǎng)的肝細(xì)胞中,CTRP9可以上調(diào)自噬水平,而自噬抑制劑3-MA可以部分抵消CTRP9對(duì)內(nèi)質(zhì)網(wǎng)應(yīng)激、凋亡和肝脂肪變性的作用。本研究擬從自噬的角度探討CTRP9在AS發(fā)生發(fā)展過(guò)程中的作用及可能機(jī)制。2研究目的(1)研究外源性CTRP9干預(yù)對(duì)動(dòng)脈粥樣硬化斑塊發(fā)生發(fā)展的影響;(2)探討CTRP9參與動(dòng)脈粥樣硬化斑塊發(fā)生發(fā)展過(guò)程的可能機(jī)制。3研究方法3.1動(dòng)脈粥樣硬化早期斑塊模型的建立8周齡雄性APoE-/-小鼠購(gòu)自北京維通利華公司,給予1周適應(yīng)性普食后改為高脂喂養(yǎng)(0.25%膽固醇和15%黃油)。4周后隨機(jī)分為3組:CTRP9慢病毒過(guò)表達(dá)組(Lv-CTRP9組),慢病毒空載體干預(yù)組(Lv-eGFP組)和PBS對(duì)照組(Control組),Lv-CTRP9組每只小鼠尾靜脈注射2×107TU攜帶CTRP9的慢病毒,Lv-eGFP組和Control組分別注射等體積的空載體慢病毒及無(wú)菌PBS。3.2小鼠血清學(xué)檢測(cè)實(shí)驗(yàn)結(jié)束后小鼠安樂(lè)死并于心尖部取血,離心后收集上層血清。ELISA法檢測(cè)血清中炎癥因子MCP-1和TNF-α的表達(dá)水平,并檢測(cè)血清中總膽固醇(TC)、甘油三酯(TG),高密度脂蛋白膽固醇(HDL-C),低密度脂蛋白膽固醇(LDL-C)的水平。3.3組織病理學(xué)檢測(cè)小鼠安樂(lè)死后,取小鼠主動(dòng)脈根部OCT包埋,以5μm厚度連續(xù)切片制作主動(dòng)脈根部冰凍切片,行HE染色觀察斑塊形態(tài),油紅O染色檢測(cè)主動(dòng)脈根部脂質(zhì)含量。同時(shí)取小鼠主動(dòng)脈行大體油紅O染色。病變的嚴(yán)重程度用總斑塊面積占相應(yīng)主動(dòng)脈面積的百分比表示。3.4免疫組織化學(xué)染色取主動(dòng)脈根部冰凍切片,免疫組織化學(xué)法檢測(cè)主動(dòng)脈根部斑塊內(nèi)巨噬細(xì)胞、平滑肌細(xì)胞的含量。Image-Pro Plus 6.0軟件計(jì)算陽(yáng)性染色面積占相應(yīng)斑塊面積的百分比。3.5 Western blot取新鮮小鼠主動(dòng)脈組織標(biāo)本,分別提取蛋白,用Western blot方法檢測(cè)MCP-1、TNF-α、LC3B、SQSTM1/p62 以及 p-AMPK、p-mTOR 等表達(dá)水平的變化。3.6統(tǒng)計(jì)學(xué)分析采用SPSS 18.0進(jìn)行統(tǒng)計(jì)學(xué)分析,所有數(shù)據(jù)均用均數(shù)±標(biāo)準(zhǔn)差表示。用單因素方差分析(ANOVA)進(jìn)行多組間統(tǒng)計(jì)分析。P0.05被認(rèn)為差異具有統(tǒng)計(jì)學(xué)意義。4結(jié)果4.1 CTRP9慢病毒轉(zhuǎn)染可以增加血清中CTRP9的表達(dá)小鼠尾靜脈慢病毒注射1周后取材,小鼠主動(dòng)脈根部斑塊內(nèi)可見GFP熒光顯著表達(dá)。Western blot結(jié)果顯示,Lv-CTRP9組血清中CTRP9蛋白表達(dá)明顯高于Lv-eGFP組與Control組(p0.05),而Lv-eGFP組和Control組未見顯著差異(p0.05)。4.2 CTRP9對(duì)小鼠血脂水平的影響Lv-CTRP9組、Lv-eGFP組小鼠和Control組三組小鼠之間血脂水平未見顯著性差異(p0.05)。4.3 CTRP9過(guò)表達(dá)可減少ApoE-/-小鼠AS斑塊面積小鼠主動(dòng)脈大體油紅O染色顯示,Lv-CTRP9組較Lv-eGFP組和Control組的主動(dòng)脈斑塊面積顯著減少(p0.05),主動(dòng)脈根部油紅O染色及HE染色結(jié)果與大體油紅O一致(p0.05)。4.4 CTRP9可以降低斑塊內(nèi)巨噬細(xì)胞浸潤(rùn)免疫組化結(jié)果顯示,Lv-CTRP9組斑塊內(nèi)巨噬細(xì)胞含量較Lv-eGFP組和Control 組降低(p0.05),而 Lv-eGFP 組和 Control 組間無(wú)明顯差異(p0.05)。4.5 CTRP9可以降低斑塊內(nèi)平滑肌細(xì)胞聚集免疫組化結(jié)果顯示,Lv-CTRP9組斑塊內(nèi)平滑肌細(xì)胞含量較Lv-eGFP組和Control 組降低(p0.05),而 Lv-eGFP 組和 Control 組間無(wú)顯著差異(p0.05)。4.6 CTRP9可以抑制ApoE-/-小鼠炎癥因子的表達(dá)Western blot結(jié)果顯示,與Lv-eGFP組和Control組相比,Lv-CTRP9組小鼠主動(dòng)脈組織內(nèi)炎癥因子MCP-1和TNF-α蛋白的表達(dá)下降(p0.05),而Lv-eGFP組和Control組間未見顯著差異(p0.05)。同時(shí),ELISA結(jié)果顯示,與兩對(duì)照組相比,CTRP9可以降低血清中MCP-1和TNF-α的表達(dá)水平(p0.05),而兩對(duì)照組間無(wú)顯著差異(p0.05)。4.7 CTRP9對(duì)ApoE--小鼠自噬水平的影響Western blot結(jié)果顯示,與Lv-eGFP組和Control組相比,Lv-CTRP9組小鼠主動(dòng)脈組織內(nèi)LC3B-Ⅱ表達(dá)增加而SQSTM1/p62表達(dá)降低(p0.05),兩對(duì)照組間未見顯著差異(p0.05)。4.8 AMPK/mTOR信號(hào)通路介導(dǎo)了 CTRP9對(duì)自噬水平的調(diào)節(jié)Western blot 結(jié)果顯示,與 Lv-eGFP 組和 Control 組相比,Lv-CTRP9 組 AMPK磷酸化水平增加(p0.05),mTOR磷酸化水平降低(p0.05),而Lv-eGFP組和Control組間AMPK及mTOR的磷酸化水平未見顯著差異(p0.05)。5結(jié)論(1)CTRP9過(guò)表達(dá)可抑制動(dòng)脈粥樣硬化斑塊發(fā)生發(fā)展;(2)CTRP9過(guò)表達(dá)可增加動(dòng)脈粥樣硬化斑塊內(nèi)自噬水平;(3)CTRP9的抗動(dòng)脈粥樣硬化作用可能與AMPK/mTOR信號(hào)通路介導(dǎo)的自噬相關(guān)。
[Abstract]:1 research background cardio cerebral vascular disease is the most important cause of death in the world. The pathological basis is atherosclerosis (AS). A large number of studies have proved that AS is a major chronic inflammatory and metabolic disease involved in the large and medium vessel wall, the pathophysiological process of.AS is very complicated, and its pathogenesis is present. Autophagy is still needed to be further clarified. Autophagy is a highly conserved process in which eukaryotes are highly conserved in the process of lysosome degradation of their own organelles and proteins. With the progress of the study, autophagy is considered to play an important role in the development of AS, and a variety of AS risk factors can cause autophagy. Disorder. Autophagy can regulate oxidative stress, metabolism, inflammation and apoptosis in AS. Moderate level of autophagy may play a protective role in AS, so regulation of autophagy may be an important target for the treatment of atherosclerosis related cardiovascular diseases. Mammal reamamycin target protein (the mammalian target of rapamycin, mT) OR) is the target molecule of rapamycin in mammalian cells. MTOR has two complexes of mTORC1 and mTORC2 in the cells, in which mTORC1 participates in the regulation of the growth and autophagy of many cells. The inhibition of mTORC1 can play the role of AS protection in a variety of ways, such as improving endothelial dysfunction, inhibiting monocyte adhesion and aggregation, and reducing the concentration of mononuclear cells. Adenosine monophosphate-activated protein kinase (AMPK), an important energy receptor of eukaryotic cells, can negatively regulate the activity of mTORC1 and up regulation of autophagic water. More and more evidence shows that.C1q/ tumor necrosis factor related protein 9 (C1q/TNF-relatedprotein9, CTRP9), a new target for activating AMPK or inhibiting mTOR or atherosclerosis, is a new type of adipocytokine in the CTRP superfamily, highly homologous with adiponectin (adiponectin, APN). The study shows that CTRP9 is in the regulation of metabolism and protection. Myocardium, improvement of endothelial function and resistance to cardiovascular damage in diabetes. More and more studies have shown that the protective effect of CTRP9 on cardiovascular system.CTRP9 can retard vascular remodeling after vascular injury by regulating the proliferation and migration of smooth muscle cells. At the same time, CTRP9 can inhibit the heart by inhibiting the heart. Myocyte apoptosis and inflammatory response reduce myocardial damage. In addition, serum CTRP9 levels can be used as a predictor of APN independent predictors of cardiovascular events. Our previous study showed that the serum levels of CTRP9 and APN in patients with acute coronary syndrome (acute coronary syndrome, ACS) decreased, and CT, compared with healthy controls, and CT The serum levels of RP9 and APN are negatively correlated with the severity of ACS lesions. We further found that CTRP9 can increase the stability of AS vulnerable plaque in the AS vulnerable plaque model mice. Recent studies have shown that CTRP9 can increase the autophagy level in human primary cultured hepatocytes, and the autophagy inhibitor 3-MA can partially counteract the CTRP9 to the endoplasmic reticulum. The effect of excitation, apoptosis and hepatic steatosis. The purpose of this study is to explore the role of CTRP9 in the development of AS and the possible mechanism of.2 (1) to study the effect of exogenous CTRP9 intervention on the development of atherosclerotic plaque, and (2) to explore the possible mechanism of CTRP9 participation in the development of atherosclerotic plaques. 3 study method 3.1 atherosclerotic early plaque model established 8 weeks old male APoE-/- mice bought from Beijing vitamin a company, given 1 weeks of adaptive diet to high fat feeding (0.25% cholesterol and 15% butter) after.4 weeks, randomly divided into 3 groups: CTRP9 lentivirus overexpression group (Lv-CTRP9 group), lentivirus airborne intervention group (Lv-eGFP group) and PBS control group (group Control), group Lv-CTRP9 each mouse tail vein injection of 2 x 107TU to carry CTRP9 of the lentivirus, Lv-eGFP and Control groups were injected with equal volume of no-load lentivirus and aseptic PBS.3.2 mice after serological test after the end of euthanasia and blood from the tip of the heart, centrifuged to collect the upper serum.ELISA method to detect serum The expression level of medium inflammatory factors MCP-1 and TNF- a, and detection of serum total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C),.3.3 histopathology test mice after euthanasia, take the aorta root OCT embedded in the aorta, and make the aorta root with 5 u thickness continuous slice. The morphology of the plaque was observed by HE staining. The lipid content of the aortic root was detected by oil red O staining. The aorta of the aorta was stained with gross oil red O. The severity of the lesion was measured by the percentage of the total patch area of the corresponding aorta. The frozen section of the aorta root was obtained by.3.4 immunohistochemical staining, and the immunohistochemical method was used. The content of macrophages and smooth muscle cells in the aortic root plaque was detected by.Image-Pro Plus 6, the percentage of the positive staining area was calculated as a percentage of the corresponding patch area.3.5 Western blot for the fresh mouse aortic tissue samples, and the protein was extracted respectively. Western blot method was used to detect MCP-1, TNF- alpha, LC3B, SQSTM1/p62, and p-AMPK. .3.6 statistical analysis of SPSS 18 was used for statistical analysis. All data were represented by mean mean deviation. Statistical analysis of multiple groups by single factor variance analysis (ANOVA).P0.05 was considered to have statistical significance.4 results and 4.1 CTRP9 lentivirus transfection could increase the expression of CTRP9 in serum in mice. 1 weeks after intravenous injection of lentivirus, the results of.Western blot in the aortic root plaque showed that the expression of CTRP9 protein in the serum of the Lv-CTRP9 group was significantly higher than that of the Lv-eGFP group and the Control group (P0.05), but there was no significant difference between the Lv-eGFP group and the Control group (P0.05) and the effect of.4.2 Lv-CTRP9 on the blood lipid level of mice was found. There was no significant difference in blood lipid levels between the three groups of group Lv-eGFP mice and group Control (P0.05).4.3 CTRP9 overexpression could reduce the aorta red O staining in the aorta of ApoE-/- mice and the aorta plaque surface accumulation in the Lv-CTRP9 group was significantly lower than that of the Lv-eGFP and Control groups (P0.05), and the aorta root was stained with oil red staining and The results of HE staining with gross oil red O (P0.05).4.4 CTRP9 could reduce the immuno histochemical results of macrophage infiltration in plaque, which showed that the content of macrophages in the Lv-CTRP9 group was lower than that in the Lv-eGFP group and Control group (P0.05), but there was no significant difference between the Lv-eGFP and Control groups (P0.05) could reduce the smooth muscle cells in the plaque. The results of aggregation immunohistochemistry showed that the content of smooth muscle cells in Lv-CTRP9 group was lower than that in group Lv-eGFP and Control group (P0.05), but there was no significant difference between group Lv-eGFP and Control group (P0.05).4.6 CTRP9 could inhibit the expression of inflammatory factors in ApoE-/- mice. The expression of inflammatory factors MCP-1 and TNF- alpha protein in the aorta decreased (P0.05), but there was no significant difference between the Lv-eGFP group and the Control group (P0.05). At the same time, the ELISA results showed that CTRP9 could reduce the level of MCP-1 and TNF- alpha in the serum (P0.05) compared with the two control group, but there was no significant difference between the two control groups (P0.05) The effect of Western blot on the autophagy level in mice showed that the expression of LC3B- II in the aorta of the Lv-CTRP9 group increased and the SQSTM1/p62 expression decreased (P0.05) compared with the Lv-eGFP group and the Control group, and there was no significant difference between the two control groups (P0.05).4.8 AMPK/mTOR signaling pathway mediated the regulation of the regulation of the level of autophagy. Compared with group Lv-eGFP and group Control, the level of AMPK phosphorylation in Lv-CTRP9 group increased (P0.05), mTOR phosphorylation level decreased (P0.05), but there was no significant difference in the phosphorylation level of AMPK and mTOR between Lv-eGFP and Control groups (P0.05) conclusion (1) overexpression could inhibit the development of atherosclerotic plaques; (2) overexpression may be overexpressed. Increased autophagy level in atherosclerotic plaques; (3) the anti atherogenic effect of CTRP9 may be related to autophagy mediated by AMPK/mTOR signaling pathway.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R543.5

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 李海云，王公瑞;大白鼠主動(dòng)脈剪切特性的試驗(yàn)研究[J];重慶大學(xué)學(xué)報(bào)(自然科學(xué)版);1994年02期

2 陳瑛;藺莉;金華;;絕經(jīng)早期和絕經(jīng)晚期小鼠主動(dòng)脈病理改變及雌激素受體表達(dá)的對(duì)比研究[J];中國(guó)全科醫(yī)學(xué);2010年30期

3 嚴(yán)宗毅;;主動(dòng)脈零應(yīng)力狀態(tài)與種屬的關(guān)系:豬與鼠[J];國(guó)外醫(yī)學(xué).生物醫(yī)學(xué)工程分冊(cè);1992年02期

4 張莉;吳賽珠;張帆;洪雷;賴文巖;;生理劑量的雄激素對(duì)去勢(shì)小鼠主動(dòng)脈線粒體DNA缺失突變的影響[J];南方醫(yī)科大學(xué)學(xué)報(bào);2011年01期

5 唐志剛;;大白鼠主動(dòng)脈脂代謝中的衰老作用[J];國(guó)外醫(yī)學(xué)(老年醫(yī)學(xué)分冊(cè));1986年03期

6 張莉;吳賽珠;阮云軍;張帆;洪雷;劉公理;王琦;;雄激素缺乏對(duì)雄性小鼠主動(dòng)脈增齡性變化的影響[J];熱帶醫(yī)學(xué)雜志;2011年02期

7 歷志;魏旭峰;顧春虎;康曉軍;王春梅;韓驊;易定華;;正常小鼠主動(dòng)脈瓣膜的組織形態(tài)學(xué)研究[J];心臟雜志;2010年05期

8 趙書民,朱琳,李利軍;小鼠主動(dòng)脈發(fā)生的觀察[J];解剖學(xué)報(bào);1997年01期

9 周信;張小榮;張秋燕;崔翰明;;生半夏及其炮制品對(duì)小鼠主動(dòng)脈內(nèi)皮細(xì)胞炎性因子分泌的影響[J];中國(guó)實(shí)驗(yàn)方劑學(xué)雜志;2013年10期

10 T.Izumi;速凍、深蝕刻復(fù)制顯示鼠主動(dòng)脈內(nèi)皮細(xì)胞小泡的胞漿表面膜的紋狀結(jié)構(gòu)[J];廣東解剖學(xué)通報(bào);1989年02期

相關(guān)會(huì)議論文 前2條

1 仲英潔;張子為;徐郁;花曉敏;胡云;;阿托伐他汀對(duì)ApoE~(-/-)小鼠主動(dòng)脈粥樣硬化和血管鈣化的影響及機(jī)制研究[A];中華醫(yī)學(xué)會(huì)糖尿病學(xué)分會(huì)第十六次全國(guó)學(xué)術(shù)會(huì)議論文集[C];2012年

2 楊婭;王艷紅;李治安;謝謹(jǐn)捷;秦彥文;;正常小鼠主動(dòng)脈的超聲生物顯微鏡顯像分析[A];中華醫(yī)學(xué)會(huì)第十次全國(guó)超聲醫(yī)學(xué)學(xué)術(shù)會(huì)議論文匯編[C];2009年

相關(guān)博士學(xué)位論文 前1條

1 齊巖梅;神經(jīng)生長(zhǎng)因子對(duì)小鼠主動(dòng)脈環(huán)血管新生及人臍靜脈內(nèi)皮細(xì)胞凋亡的影響[D];大連醫(yī)科大學(xué);2008年

相關(guān)碩士學(xué)位論文 前6條

1 牛榮榮;外源性磷酸肌酸對(duì)動(dòng)脈粥樣硬化斑塊形成和穩(wěn)定性的作用的研究[D];山東大學(xué);2015年

2 張炎;慢性炎癥刺激對(duì)小鼠主動(dòng)脈內(nèi)皮脂肪酶表達(dá)影響的研究[D];山東大學(xué);2016年

3 黃志廣;重組SAK-HV對(duì)小鼠主動(dòng)脈內(nèi)皮細(xì)胞的作用及機(jī)制研究[D];廣西醫(yī)科大學(xué);2016年

4 黃承敏;CTRP9對(duì)動(dòng)脈粥樣硬化斑塊進(jìn)展的作用及機(jī)制研究[D];山東大學(xué);2017年

5 張莉;去勢(shì)對(duì)小鼠主動(dòng)脈衰老的影響及生理劑量睪酮的干預(yù)研究[D];南方醫(yī)科大學(xué);2011年

6 劉永玲;外源性微直流電場(chǎng)對(duì)小鼠主動(dòng)脈環(huán)血管新生的影響[D];云南師范大學(xué);2014年

，

本文編號(hào)：1949171