动脉粥样硬化中转录及表观遗传调控巨噬细胞极化的作用及机制研究

发布时间：2018-08-03 20:47

【摘要】：AS性缺血性心、脑血管疾病凭借其高发病率、高致残率、高致死率,严重危害人类的生命健康。目前,促进AS斑块的稳定性是防治心脑血管疾病的主要方法之一。炎症反应贯穿AS的整个病理过程,炎症所介导的脂质、能量代谢异常、氧化应激、内皮损伤、细胞凋亡等直接增加了AS斑块的不稳定性,提高了急性血管事件的发生率。近年来,越来越多的研究发现,基于抗击炎症的治疗方法能够有效延缓AS的进展、减少AS性疾病的发生。巨噬细胞通过分泌大量炎性介质,如细胞因子、趋化因子、黏附分子、基质金属酶等在AS炎症反应中发挥重要作用。在不同的刺激条件下,巨噬细胞可极化为促炎亚型(M1)或抗炎亚型(M2)。近年来,通过干预巨噬细胞极化的各个环节,抑制巨噬细胞向M1亚型极化,促进巨噬细胞向M2亚型极化逐渐成为AS及AS相关疾病防治的新策略。研究证实,ox LDL是促使AS炎症反应启动和放大的重要致损因素,ox LDL能够通过诱导巨噬细胞活化为促炎的M1型,介导一系列炎症反应的发生。尽管目前已经发现一些信号通路参与调节ox LDL诱导的巨噬细胞极化,但具体机制尚未完全明确。因此,为了深入了解ox LDL影响巨噬细胞极化的可能机制,为AS性疾病的防治提供新的思路,我们进行了以下三部分研究:第一部分ox LDL对巨噬细胞极化影响的研究目的:探究ox LDL对不同种属来源巨噬细胞表型标志物及相关细胞因子表达的影响,明确ox LDL诱导巨噬细胞极化的方向。方法:(1)给予小鼠RAW264.7巨噬细胞不同浓度ox LDL处理,检测M1、M2亚型相关表型标志物、细胞因子的表达与分泌水平。(2)给予人THP1巨噬细胞不同浓度ox LDL处理,检测M1、M2亚型相关表型标志物、细胞因子的表达水平。(3)诱导冠心病、急性大动脉粥样硬化性脑梗死患者及健康供者外周血中的单个核细胞为原代巨噬细胞,给予ox LDL处理,检测M1、M2亚型相关表型标志物及细胞因子的表达水平,并观察细胞的形态变化。结果:(1)ox LDL处理后,小鼠RAW264.7巨噬细胞中M1表型标志物i NOS及M1相关细胞因子TNF-α、IL-1β、MCP1的m RNA水平与蛋白水平明显高于对照组,具有显著性差异,p0.05;小鼠M2表型标志物Arg1的m RNA水平与对照组无显著性差异,p0.05,但其蛋白水平明显低于对照组;M2相关细胞因子VEGF的表达仅在高浓度ox LDL处理时高于对照组,具有显著性差异,p0.05。(2)ox LDL处理后,人THP1巨噬细胞中i NOS及TNF-α表达明显高于对照组,具有显著性差异,p0.05;人M2表型标志物CD206及相关VEGF的表达无显著性差异,p0.05。(3)ox LDL处理后,人原代巨噬细胞中M1相关基因的表达高于对照组;CD206的表达低于对照组,但VEGF的表达高于对照组;ox LDL处理后,原代巨噬细胞形态发生明显改变。结论:ox LDL诱导不同种属来源的巨噬细胞向M1亚型而不是M2亚型极化。第二部分ox LDL通过NF-κB-HIF-1β信号通路诱导巨噬细胞极化的研究目的:通过抑制巨噬细胞中NF-κB及HIF-1β表达,探究二者在转录水平上对ox LDL诱导巨噬极化的调控作用及机制。方法:(1)采用不同时间/浓度梯度ox LDL处理小鼠RAW264.7巨噬细胞,检测NF-κB信号通路的活化水平。(2)采用PTL预处理抑制NF-κB活性后,检测ox LDL对小鼠巨噬细胞表型标志物及细胞因子表达水平的影响。(3)诱导冠心病、急性大动脉粥样硬化性脑梗死患者及健康供者外周血中的单个核细胞为原代巨噬细胞,PTL预处理后,检测ox LDL对巨噬细胞中M1、M2相关基因表达水平的影响,并观察细胞形态变化。(4)不同时间/浓度梯度ox LDL处理小鼠巨噬细胞,检测HIF家族蛋白表达水平。(5)采用sh RNA沉默小鼠巨噬细胞中HIF-1β,检测ox LDL对巨噬细胞表型标志物及细胞因子表达水平的影响。(6)分别抑制巨噬细胞中NF-κB与HIF-1β的表达,探究ox LDL条件下NF-κB与HIF-1β之间的相互调节作用。结果:(1)ox LDL处理后,小鼠巨噬细胞中NF-κB信号通路中的经典途径及非经典途径均被激活。(2)PTL预处理能够削弱ox LDL诱导的小鼠巨噬细胞中M1相关基因的表达,促进M2相关基因表达,具有显著性差异,p0.05。(3)PTL预处理能够削弱ox LDL诱导的人原代巨噬细胞中M1相关基因及VEGF的表达,促进M2相关CD206表达增加,并阻断ox LDL诱导的细胞形态改变。(4)ox LDL处理提高小鼠巨噬细胞中HIF的蛋白表达水平。(5)sh RNA沉默巨噬细胞中的HIF-1β能够削弱ox LDL诱导的M1相关基因的表达,促进M2相关基因表达升高,具有显著性差异,p0.05。(6)抑制NF-κB活化后,HIF-1β表达受抑制;沉默HIF-1β后,NF-κB活化不受影响。结论:(1)NF-κB与HIF-1β参与调控ox LDL诱导巨噬细胞向M1型极化。(2)抑制NF-κB、HIF-1β能够调控ox LDL诱导的巨噬细胞向M2亚型方向极化。(3)NF-κB位于HIF-1β上游,调控HIF-1β的表达。第三部分ox LDL通过KDM4A/JMJD2A诱导巨噬细胞极化的研究目的:通过沉默去甲基化酶KDM4A的表达,探究KDM4A在表观遗传水平上参与调控ox LDL诱导巨噬极化的机制。方法:(1)采用不同时间/浓度梯度ox LDL处理小鼠RAW264.7及THP1巨噬细胞,检测KDM4A的表达水平。(2)采用sh RNA沉默小鼠巨噬细胞中的KDM4A,检测ox LDL对巨噬细胞表型标志物及细胞因子表达水平的影响。(3)分别抑制小鼠巨噬细胞中NF-κB、HIF-1β和KDM4A的表达,探究ox LDL条件下KDM4A表达与NF-κB-HIF-1β信号通路之间的相互作用。(4)采用KDM4A抑制剂JIB-04,处理小鼠巨噬细胞,检测细胞凋亡情况,探究KDM4A在维持巨噬细胞生存中的作用。结果:(1)ox LDL处理明显增加小鼠RAW264.7及人THP1巨噬细胞KDM4A的表达水平。(2)sh RNA沉默巨噬细胞中的KDM4A能够削弱ox LDL诱导的M1相关基因的表达,促进M2相关基因表达升高,具有显著性差异,p0.05。(3)KDM4A的表达不依赖于NF-κB-HIF-1β信号通路,反之亦然。(4)JIB-04处理能够增加巨噬细胞凋亡率,ox LDL部分削弱JIB-04介导的凋亡,具有显著性差异,p0.05。结论:(1)KDM4A参与调控ox LDL诱导巨噬细胞向M1型极化。(2)KDM4A的表达与NF-κB-HIF-1β信号通路相互独立。(3)KDM4A参与调控巨噬细胞存活。
[Abstract]:AS sex ischemic heart, cerebrovascular disease, with its high incidence, high disability rate and high mortality, seriously endangers human life and health. At present, promoting the stability of AS plaque is one of the main methods to prevent and cure cardiovascular and cerebrovascular diseases. The inflammatory reaction runs through the whole pathological process of AS, the lipid metabolism, the oxidative stress, and the oxidative stress. Skin injury, apoptosis and so on directly increase the instability of AS plaque and increase the incidence of acute vascular events. In recent years, more and more studies have found that the treatment based on anti inflammation can effectively delay the progress of AS and reduce the occurrence of AS diseases. Macrophages are chemotactic by secreting a large number of inflammatory mediators, such as cytokines. Factors, adhesion molecules, matrix metalloenzyme and so on play an important role in the AS inflammatory response. In different stimuli, macrophages can polarize proinflammatory subtype (M1) or anti-inflammatory subtype (M2). In recent years, the polarization of macrophage to M1 subtype is inhibited by interfering with each link of macrophage polarization to promote the gradual polarization of macrophages to the M2 subtype. As a new strategy for the prevention and control of AS and AS related diseases, it has been confirmed that ox LDL is an important factor contributing to the initiation and amplification of AS inflammatory reactions. Ox LDL can induce a series of inflammatory reactions by inducing macrophage activation as a pro-inflammatory M1, although some signal pathways are currently found to be involved in the regulation of macrophage fines induced by ox LDL. Therefore, in order to understand the possible mechanism of ox LDL's influence on macrophage polarization, and to provide new ideas for the prevention and control of AS sex diseases, we have conducted the following three parts: the first part of the research on the effect of ox LDL on the polarization of macrophages: To explore the ox LDL for the giant macrophages of different species. The influence of the expression of cell phenotype markers and related cytokines, and to clarify the direction of ox LDL induced polarization of macrophages. Methods: (1) the RAW264.7 macrophages in mice were treated with different concentrations of ox LDL, and M1, M2 subtype related phenotypic markers were detected, the expression of cytokines and secretory water were flat. (2) the different concentration ox LDL treatment of THP1 macrophages was given. Detection of M1, M2 subtype related phenotypic markers, the expression level of cytokines. (3) induced coronary heart disease, acute large atherosclerotic cerebral infarction and healthy donor peripheral blood mononuclear cells were primary macrophages, ox LDL treatment was given to detect the expression level of phenotypic markers and cytokines of M1, M2 subtypes, and to observe the expression level. Results: (1) after ox LDL treatment, the M1 phenotype markers of RAW264.7 macrophages in mice were I NOS and M1 related cytokines TNF- a, IL-1 beta, MCP1 m RNA level and protein level were significantly higher than those of the control group, but there was no significant difference between the mice and the control group. Its protein level was significantly lower than that of the control group; the expression of M2 related cytokine VEGF was higher than that of the control group at high concentration of ox LDL. The expression of I NOS and TNF- alpha in human THP1 macrophages was significantly higher than that in the control group after p0.05. (2) ox LDL treatment. There was no significant difference. After p0.05. (3) ox LDL treatment, the expression of M1 related genes in human primary macrophages was higher than that of the control group; the expression of CD206 was lower than that of the control group, but the expression of VEGF was higher than that of the control group; the morphology of primary macrophages changed obviously after ox LDL treatment. Conclusion: ox LDL induces macrophages from different species to M1 subtype. Not M2 subtype polarization. Second part ox LDL induced polarization of macrophage through NF- kappa B-HIF-1 beta signaling pathway: by inhibiting the expression of NF- kappa B and HIF-1 beta in macrophages, the regulatory role and mechanism of the two at the transcriptional level on ox LDL induced megagocytosis are explored. Methods: (1) use different time / concentration gradient ox LDL to treat small LDL. Mouse RAW264.7 macrophages were used to detect the activation level of NF- kappa B signaling pathway. (2) the effects of ox LDL on the phenotype markers of macrophages and the expression of cytokines in mice were detected by PTL pretreatment to inhibit NF- kappa B activity. (3) induced coronary heart disease, acute large atherosclerotic cerebral infarction and healthy donors in peripheral blood mononuclear cells The cells were primary macrophages. After PTL pretreatment, the effect of ox LDL on the expression level of M1 and M2 related genes in macrophages was detected and the morphological changes were observed. (4) the expression level of HIF family protein was detected by different time / concentration gradient ox LDL to detect the expression level of HIF family protein. (5) sh RNA was used to silence the HIF-1 beta in murine macrophages. The effects of phagocytic phenotypic markers and expression level of cytokines. (6) inhibition of the expression of NF- kappa B and HIF-1 beta in macrophages, and to explore the interaction between NF- kappa B and HIF-1 beta in ox LDL. Results: (1) the classical pathway and non classical pathway of NF- kappa B signal pathway in mouse macrophages were activated (2) PTL pretreatment can weaken the expression of M1 related genes in mouse macrophages induced by ox LDL, promote the expression of M2 related genes, and have significant differences. P0.05. (3) PTL pretreatment can weaken the expression of M1 related genes and VEGF in ox LDL induced human primary macrophages, promote the increase of M2 related expressions and block the cells induced by ox. Morphological changes. (4) ox LDL treatment enhanced the protein expression level of HIF in mouse macrophages. (5) HIF-1 beta in SH RNA silenced macrophages could weaken the expression of M1 related genes induced by ox LDL, promote the increase of the expression of M2 related genes, and have significant difference. P0.05. (6) inhibited the activation of NF- kappa. - kappa B activation is not affected. Conclusion: (1) NF- kappa B and HIF-1 beta participate in the regulation of ox LDL induced macrophages to M1 polarization. (2) inhibition of NF- kappa B, HIF-1 beta can regulate the orientation of macrophages induced by ox LDL. (3) Objective: To explore the mechanism of KDM4A at the epigenetic level to regulate ox LDL induced macrophage polarization through the expression of silencing demethylation enzyme KDM4A. Methods: (1) to use different time / concentration gradient ox LDL to treat mice RAW264.7 and THP1 macrophages and detect the level of KDM4A. (2) K in SH RNA murine macrophages DM4A, detect the effect of ox LDL on the expression of macrophage phenotypic markers and cytokines. (3) inhibit the expression of NF- kappa B, HIF-1 beta and KDM4A in mouse macrophages, and explore the interaction between KDM4A expression and NF- kappa B-HIF-1 beta signaling pathway in ox LDL conditions. (4) use the inhibitor to treat murine macrophages and detect the details. To explore the role of KDM4A in maintaining the survival of macrophages. Results: (1) ox LDL treatment significantly increased the expression level of KDM4A in mouse RAW264.7 and human THP1 macrophages. (2) KDM4A in SH RNA silent macrophages can weaken the expression of M1 related genes induced by ox LDL, and promote the increased expression of related genes, which has a significant difference. The expression of p0.05. (3) KDM4A is not dependent on the NF- kappa B-HIF-1 beta signaling pathway, and vice versa. (4) JIB-04 treatment can increase the apoptosis rate of macrophages, and ox LDL partially weakens JIB-04 mediated apoptosis. There is a significant difference in p0.05. conclusion: (1) KDM4A participates in the regulation of ox LDL to induce macrophage to polarization polarization. (2) Signal pathway is independent of each other. (3) KDM4A participates in regulating macrophage survival.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R543.5

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