CTRP9对动脉粥样硬化斑块进展的作用及机制研究

发布时间：2018-05-29 02:36

本文选题：Clq/肿瘤坏死因子相关蛋白9 + 动脉粥样硬化　；参考：《山东大学》2017年硕士论文

【摘要】：1研究背景心脑血管疾病是目前世界范围内致残、致死的首位原因,其病理基础主要是动脉粥样硬化(atherosclerosis,AS)。大量研究已证实AS是一种主要累及大中血管壁的慢性炎症和代谢性疾病。AS的病理生理过程十分复杂,目前其发病机制仍有待于进一步阐明。自噬是真核生物在进化过程中高度保守的依赖溶酶体降解细胞内自身细胞器和蛋白质的自我保护过程。随着研究的进展,目前认为自噬在AS发生发展过程中扮演着重要的角色,多种AS危险因素均可引起自噬功能紊乱。自噬在AS中可以参与调节氧化应激、代谢、炎症和凋亡等过程。适度水平的自噬对AS起保护性作用,因此调节自噬水平或许能成为治疗动脉粥样硬化相关性心血管疾病的重要靶点。哺乳动物雷帕霉素靶蛋白(the mammalian target of rapamycin,mTOR)是雷帕霉素在哺乳动物细胞内的靶分子,mTOR在细胞内存在mTORC1和mTORC2两种复合体,其中mTORC1参与调节多种细胞的生长和自噬等过程。抑制mTORC1可以通过多种途径发挥AS保护作用,如改善内皮功能紊乱、抑制单核细胞粘附和聚集、减少斑块内泡沫细胞形成、降低巨噬细胞炎症反应以及延缓平滑肌细胞增殖和迁移等。腺苷酸活化蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)作为真核细胞的一个重要能量感受器,活化后可以负性调节mTORC1的活性,从而上调自噬水平。越来越多的证据显示,活化AMPK或者抑制mTOR或为动脉粥样硬化防治的新靶点。C1q/肿瘤坏死因子相关蛋白 9(C1q/TNF-relatedprotein9,CTRP9)是 CTRP超家族中的新型脂肪细胞因子,与脂联素(adiponectin,APN)高度同源。研究显示CTRP9在调节代谢、保护心肌、改善内皮功能及抵抗糖尿病心血管损害等方面发挥重要作用。越来越多的研究展现出了 CTRP9对心血管系统的保护性作用。CTRP9可以通过调控平滑肌细胞增殖和迁移过程延缓血管损伤后病理性血管重塑。同时,CTRP9可以通过抑制心肌细胞凋亡和炎症反应降低心肌损伤。此外,血清CTRP9水平可作为一个独立于APN的预测因素评估心血管事件的发生。我们前期研究显示,与健康对照人群相比,急性冠脉综合症(acute coronary syndrome,ACS)患者血清中CTRP9和APN水平均降低,并且CTRP9和APN的血清水平与ACS病变严重程度呈负相关。我们进一步发现在AS易损斑块模型小鼠中,CTRP9可增加AS易损斑块的稳定性。近期研究显示,在人原代培养的肝细胞中,CTRP9可以上调自噬水平,而自噬抑制剂3-MA可以部分抵消CTRP9对内质网应激、凋亡和肝脂肪变性的作用。本研究拟从自噬的角度探讨CTRP9在AS发生发展过程中的作用及可能机制。2研究目的(1)研究外源性CTRP9干预对动脉粥样硬化斑块发生发展的影响;(2)探讨CTRP9参与动脉粥样硬化斑块发生发展过程的可能机制。3研究方法3.1动脉粥样硬化早期斑块模型的建立8周龄雄性APoE-/-小鼠购自北京维通利华公司,给予1周适应性普食后改为高脂喂养(0.25%胆固醇和15%黄油)。4周后随机分为3组:CTRP9慢病毒过表达组(Lv-CTRP9组),慢病毒空载体干预组(Lv-eGFP组)和PBS对照组(Control组),Lv-CTRP9组每只小鼠尾静脉注射2×107TU携带CTRP9的慢病毒,Lv-eGFP组和Control组分别注射等体积的空载体慢病毒及无菌PBS。3.2小鼠血清学检测实验结束后小鼠安乐死并于心尖部取血,离心后收集上层血清。ELISA法检测血清中炎症因子MCP-1和TNF-α的表达水平,并检测血清中总胆固醇(TC)、甘油三酯(TG),高密度脂蛋白胆固醇(HDL-C),低密度脂蛋白胆固醇(LDL-C)的水平。3.3组织病理学检测小鼠安乐死后,取小鼠主动脉根部OCT包埋,以5μm厚度连续切片制作主动脉根部冰冻切片,行HE染色观察斑块形态,油红O染色检测主动脉根部脂质含量。同时取小鼠主动脉行大体油红O染色。病变的严重程度用总斑块面积占相应主动脉面积的百分比表示。3.4免疫组织化学染色取主动脉根部冰冻切片,免疫组织化学法检测主动脉根部斑块内巨噬细胞、平滑肌细胞的含量。Image-Pro Plus 6.0软件计算阳性染色面积占相应斑块面积的百分比。3.5 Western blot取新鲜小鼠主动脉组织标本,分别提取蛋白,用Western blot方法检测MCP-1、TNF-α、LC3B、SQSTM1/p62 以及 p-AMPK、p-mTOR 等表达水平的变化。3.6统计学分析采用SPSS 18.0进行统计学分析,所有数据均用均数±标准差表示。用单因素方差分析(ANOVA)进行多组间统计分析。P0.05被认为差异具有统计学意义。4结果4.1 CTRP9慢病毒转染可以增加血清中CTRP9的表达小鼠尾静脉慢病毒注射1周后取材,小鼠主动脉根部斑块内可见GFP荧光显著表达。Western blot结果显示,Lv-CTRP9组血清中CTRP9蛋白表达明显高于Lv-eGFP组与Control组(p0.05),而Lv-eGFP组和Control组未见显著差异(p0.05)。4.2 CTRP9对小鼠血脂水平的影响Lv-CTRP9组、Lv-eGFP组小鼠和Control组三组小鼠之间血脂水平未见显著性差异(p0.05)。4.3 CTRP9过表达可减少ApoE-/-小鼠AS斑块面积小鼠主动脉大体油红O染色显示,Lv-CTRP9组较Lv-eGFP组和Control组的主动脉斑块面积显著减少(p0.05),主动脉根部油红O染色及HE染色结果与大体油红O一致(p0.05)。4.4 CTRP9可以降低斑块内巨噬细胞浸润免疫组化结果显示,Lv-CTRP9组斑块内巨噬细胞含量较Lv-eGFP组和Control 组降低(p0.05),而 Lv-eGFP 组和 Control 组间无明显差异(p0.05)。4.5 CTRP9可以降低斑块内平滑肌细胞聚集免疫组化结果显示,Lv-CTRP9组斑块内平滑肌细胞含量较Lv-eGFP组和Control 组降低(p0.05),而 Lv-eGFP 组和 Control 组间无显著差异(p0.05)。4.6 CTRP9可以抑制ApoE-/-小鼠炎症因子的表达Western blot结果显示,与Lv-eGFP组和Control组相比,Lv-CTRP9组小鼠主动脉组织内炎症因子MCP-1和TNF-α蛋白的表达下降(p0.05),而Lv-eGFP组和Control组间未见显著差异(p0.05)。同时,ELISA结果显示,与两对照组相比,CTRP9可以降低血清中MCP-1和TNF-α的表达水平(p0.05),而两对照组间无显著差异(p0.05)。4.7 CTRP9对ApoE--小鼠自噬水平的影响Western blot结果显示,与Lv-eGFP组和Control组相比,Lv-CTRP9组小鼠主动脉组织内LC3B-Ⅱ表达增加而SQSTM1/p62表达降低(p0.05),两对照组间未见显著差异(p0.05)。4.8 AMPK/mTOR信号通路介导了 CTRP9对自噬水平的调节Western blot 结果显示,与 Lv-eGFP 组和 Control 组相比,Lv-CTRP9 组 AMPK磷酸化水平增加(p0.05),mTOR磷酸化水平降低(p0.05),而Lv-eGFP组和Control组间AMPK及mTOR的磷酸化水平未见显著差异(p0.05)。5结论(1)CTRP9过表达可抑制动脉粥样硬化斑块发生发展;(2)CTRP9过表达可增加动脉粥样硬化斑块内自噬水平;(3)CTRP9的抗动脉粥样硬化作用可能与AMPK/mTOR信号通路介导的自噬相关。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R543.5

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