SOCS3在心肌细胞缺氧复氧中的作用及调控机制

发布时间：2018-04-24 12:32

本文选题：缺氧复氧 + SOCS-3　；参考：《南昌大学》2017年博士论文

【摘要】：背景:缺血性心脏病(ischemic heart disease,IHD)是一种常见的危害人类健康的疾病,仅2012年一年死亡人数就达740万。并且,IHD耗费医疗资源惊人,通过溶栓、PCI等治疗手段尽快恢复冠状动脉血流供应是挽救患者生命的关键,但研究发现心肌缺血持续一段时间后,重新恢复血流灌注会给心脏组织带来新的损伤,出现心肌顿抑、心功能降低及恶性心律失常发作等继发性心肌损害——缺血再灌注损伤(ischemia-reperfusion injury,IRI)。研究心脏缺血再灌注损伤的发生机制,探讨有效的IRI心肌保护措施长期以来都是心血管病医生孜孜以求的梦想。目前认为,心脏缺血再灌注损伤与心肌线粒体能量代谢障碍、细胞内钙超载、氧自由基释放和细胞凋亡等有关。细胞因子信号转导抑制因子(suppressor of cytokine signaling,SOCS)家族是特征性SOCS盒胞浆蛋白分子家族,包括SH2核心结构及具有保守性的C端40个氨基酸模块。该家族存在多种细胞内并可被诱导表达。目前发现该家族有CIS(the cytokine-inducible SH2 domain-containing protein)、SOCS1-7等8个成员分子。SOCS通过竞争结合JAK催化位点从而抑制JAK信号通路。其中SOCS1与SOCS3同源性最强,与心血管疾病关系最为密切。既往研究发现SOCS1竞争心肌营养素-1(cardiotrophin-1,CT-1)下游信号通路JAK的催化位点,从而抑制JAK-STAT3信号转导通路,导致心肌细胞凋亡增加及心肌梗死面积扩大。Micro RNA(miRNA)是一类内生的、长度约为18~25个核苷酸的小RNA,通过对靶m RNA降解或者翻译抑制在转录后水平发挥对靶基因负调控作用。参与细胞内多种重要调节。研究发现miRNA参与了心脏发育、心肌肥厚及重塑、心律失常、心力衰竭及心肌细胞凋亡等病理生理过程。在心肌成纤维细胞中,miR-19a通过调控TGF-βR II抑制自噬。又有报道miR-19与心肌细胞凋亡有关;最近研究发现miR-19b在心脏缺血再灌注损伤中通过调控PTEN抑制心肌细胞凋亡。鉴于miR-19a与miR-19b同源于miR-17-92基因簇,miR-19a极有可能参与心脏缺血再灌注损伤。最近又有研究发现,在肝细胞中miR-19a靶向抑制SOCS3表达增加JAK-STAT的转录并促进肝细胞增殖。由此我们推测,在缺血再灌注损伤过程中,miR-19a可能通过靶向调控SOCS3抑制心肌细胞凋亡。综上所述,我们推测在心肌IRI过程中可能存在的一个调控过程:心肌IRI时保护性细胞因子(比如CT-1等)表达上调,诱导JAK-STAT3活性增强,导致STAT3磷酸化活性增强可以上调SOCS3表达,SOCS3反过来竞争结合JAK并抑制其表达,抵消一部分STAT3对心肌细胞抗凋亡作用。如果miR-19a能抑制SOCS3的表达,间接提高JAK-STAT3的活性并增强在IRI中的抗凋亡作用。为了证实这一推测。本研究进行一下三方面的探索:首先,通过离体实验探讨SOCS3在缺血再灌注损伤中的促凋亡作用,将建立心肌细胞缺氧复氧模型,通过细胞存活率检查、生化检测等体外实验证实SOCS3与促凋亡蛋白Bim之间的关系。其次,在离体实验探讨miR-19a对SOCS3靶向调控作用机制。通过miR-19a过表达及抑制实验检测SOCS3的蛋白表达以及对心肌细胞凋亡的影响。第三,为探讨SOCS-3与急性心肌梗死具有相关性,以急性心肌梗死患者作为研究对象,分析高表达SOCS3是否可以预测心脏主要不良事件。第一部分探讨在心肌细胞缺氧复氧过程中SOCS3与Bim之间的关系及对细胞凋亡的影响目的:建立H9c2心肌细胞缺氧复氧模型,明确SOCS3在心肌细胞缺氧复氧中对Bim表达影响及细胞凋亡的影响。方法:1)培养H9c2心肌细胞。2)模拟缺血再灌注损伤建立缺氧复氧(Hypoxia reoxygenation,HR)模型,Western-blot检测SOCS3表达。3)设计与合成3对靶向SOCS3基因的si RNA(SOCS3-si RNA),Western-blot筛选最佳SOCS3-si RNA。4)将效果最佳SOCS3-si RNA转染H9c2,给予缺氧复氧。实验分4组:空白对照组(Control组)、HR组、HR+Negative Control组、HR+SOCS3-si RNA组;5)用CCK8法检测细胞增殖活力,Flow cytometry检测细胞凋亡率,Western-blot技术分别测定SOCS3、凋亡相关基因Bim、Bax和Bcl-2凋亡蛋白的表达。结果:1)缺氧复氧模过程中SOCS3表达明显上调。2)与空白对照组相比,HR组细胞存活率明显减少(P0.05),SOCS3-si RNA组H9c2存活率明显增加(P0.05),凋亡率明显下降(P0.05);3)与HR组相比,SOCS3-si RNA+HR组Bim表达下调(P0.01);4)与HR组相比,SOCS3-si RNA+HR组,Bcl-2表达上调,Bax表达下调,Bcl-2/Bax比值增大(P0.05)。结论:1)H9c2心肌细胞在缺氧复氧后,SOCS3表达明显升高;2)在心肌细胞缺氧复氧模型中,沉默SOCS3可以下调Bim和Bax的表达;3)在心肌细胞在缺氧复氧模型中,沉默SOCS3可以上调Bcl-2的表达4)在心肌细胞在缺氧复氧模型中,沉默SOCS3可以升高细胞活力,减少细胞凋亡。第二部分:缺氧复氧心肌细胞中SOCS3对JAK-STAT3信号通路的影响及miR-19a对其调控作用目的:本研究拟建立H9c2心肌细胞HR模型,测定miR-19a在HR过程中表达,在H9c2中转染miR-19a mimics或miR-19a inhibitor调控miR-19a的表达,测定细胞SOCS3的表达及细胞凋亡率、凋亡相关基因蛋白表达水平,探讨miR-19a在心肌细胞缺氧复氧中对SOCS3调控作用及介导HR细胞凋亡的影响。方法:1)模拟缺血再灌注损伤建立HR模型,q RT-PCR检测miR-19a表达。2)使用Lipofectamine3000分别将miR-19a mimics及miR-19a inhibitor转染入H9c2细胞,给予缺氧复氧。3)实验分6组:空白对照组(Control组)、HR、HR+mimics Negative Control、HR+miR-19a mimics组、HR+Mirco RNA inhibitor Negative Control组、HR+miR-19a inhibitor组;4)CCK8检测细胞活力,Flow cytometry检测细胞凋亡率,Western-blot技术分别测定SOCS3、p-STAT3、STAT3、Caspases、Bim、Bax和Bcl-2的表达。结果:1)HR过程中miR-19a表达明显下调。2)HR组较control组细胞存活率明显减少(P0.05),miR-19a mimics组H9c2细胞存活率明显增加(P0.05),凋亡率明显下降(P0.05);miR-19a inhibitor组H9c2细胞存活率明显下降(P0.05),凋亡率明显增加(P0.05);3)过表达miR-19a明显抑制SOCS3、Caspases、Bim及Bax蛋白表达,上调p-STAT3活性及Bcl-2蛋白,抑制细胞凋亡。4)抑制miR-19a明显上调SOCS3、Caspases、Bim及Bax蛋白表达,抑制 p-STAT3活性,Bcl-2表达下调,促进凋亡。结论:1)H9c2心肌细胞缺氧复氧后,miR-19a表达明显下调;2)心肌细胞缺氧复氧过程中,miR-19a可能通过调控SOCS3发挥抗凋亡作用;第三部分血浆SOCS-3上调与急性心肌梗死不良预后相关性分析目的:人血浆SOCS-3上调与急性心肌梗死不良预后相关性分析方法:入选2016.3-2016.10在我院就诊急性心肌梗死(AMI)患者(n=68),同期选择年龄、性别等相匹配的,无心血管系统疾病的自愿者作为健康对照组(n=20)。采用酶联免疫(ELISA)测定SOCS3、c Tn I和CK-MB。并将SOCS3与c Tn I、CK-MB、NT-pro BNP、e GFR、左室射血分数(left ventricular ejection fraction LVEF)、冠状动脉病变血管数、Killip心功能分级和主要不良心血管事件(MACE)等行相关分析。结果:AMI患者血浆SOCS-3表达水平显著高于健康对照组(P0.01);通过ROC曲线比较,血浆SOCS-3区分AMI患者的敏感性和特异性分别为70.0和85.5(AUC=0.856)。SOCS-3水平与高血压(P=0.002),左心室射血分数(LVEF)(P=0.004),e GFR(P0.001),c Tn I(P0.001),CK-MB(P0.001),NT-pro BNP(P0.001)等临床病理参数显著相关。此外,AMI患者冠状动脉狭窄数目越多,血浆SOCS-3水平越高。SOCS-3水平与Killip心功能分级相关,Killip心功能分级越高的患者血浆SOCS-3水平越高。AMI患者中,根据两分位中位数,高血浆SOCS-3比低血浆SOCS-3患者具有更高主要不良心血管事件(MACE)发生率(P0.01)。同样,高血浆SOCS-3较低血浆SOCS-3患者,6个月总体存活率更低(P0.01)。血浆SOCS3是MACE的独立危险因素。结论:SOCS-3是预测急性心肌梗死预后有价值的生物学标指标。总之,从分子、细胞和在体多层面证实通过抑制SOCS3的表达减轻缺血再灌注心肌损伤,为深入理解SOCS3的调控机制进而对心肌保护机制做有益的探索。
[Abstract]:Background: ischemic heart disease (IHD) is a common disease that endangers human health. The number of deaths in one year is 7 million 400 thousand in 2012 alone. And, IHD has a surprising number of medical resources. By thrombolytic and PCI treatment, the key to restore the blood flow of the coronary artery as soon as possible is the key to save the life of the patients, but the study found myocardial deficiency. After a period of blood continuous time, a new recovery of blood perfusion will bring new damage to the heart tissue, and secondary myocardial damage, such as myocardial stunning, cardiac dysfunction and malignant arrhythmia attack, such as ischemia-reperfusion injury (IRI). The mechanism of myocardial ischemia reperfusion injury is studied, and the effective IRI is explored. Myocardial protection has long been the dream of cardiovascular doctors. It is believed that myocardial ischemia reperfusion injury is related to myocardial mitochondrial energy metabolism disorder, intracellular calcium overload, oxygen free radical release and cell apoptosis. Cytokine signal transduction inhibitor (suppressor of cytokine signaling, SOCS) family is The characteristic SOCS box cytoplasmic protein family, including the SH2 core structure and the conserved C terminal 40 amino acid modules. The family exists in a variety of cells and can be induced to be induced. At present, the family has CIS (the cytokine-inducible SH2 domain-containing protein), and SOCS1-7 and other 8 members are.SOCS through competition and JAK. Inhibition of JAK signaling pathways. Among them, SOCS1 and SOCS3 have the strongest homology and are most closely related to cardiovascular disease. Previous studies have found that SOCS1 competitive cardiomyotrophic -1 (cardiotrophin-1, CT-1) downstream signal pathway JAK's catalytic site, thus inhibiting the JAK-STAT3 signal transduction pathway, leading to increased cardiomyocyte apoptosis and myocardial infarction Area enlargement of.Micro RNA (miRNA) is a class of endogenous, small RNA with a length of about 18~25 nucleotides. By degrading the target m RNA or suppressing the negative regulation of the target gene at post transcriptional levels, it participates in a variety of important intracellular regulations. The study found miRNA involved in heart development, cardiac hypertrophy and remodeling, arrhythmia, and heart failure. The pathophysiological processes such as exhaustion and cardiomyocyte apoptosis. In myocardial fibroblasts, miR-19a inhibits autophagy by regulating TGF- beta R II. It is also reported that miR-19 is associated with cardiomyocyte apoptosis; recent studies have found that miR-19b inhibits cardiomyocyte apoptosis by regulating PTEN in ischemic reperfusion injury. In view of miR-19a and miR-19b homologous to miR-17 -92 gene cluster, miR-19a may be very likely to participate in the ischemic reperfusion injury of the heart. Recently, it has been found that the expression of miR-19a targeting inhibition of SOCS3 in hepatocytes increases the transcription of JAK-STAT and promotes the proliferation of hepatocytes. Thus, we speculate that in the course of ischemia reperfusion injury, miR-19a can inhibit the apoptosis of cardiac myocytes through the targeting regulation of SOCS3. In summary, we speculate that a regulatory process may exist during the IRI process in the myocardium: the expression of protective cytokines (such as CT-1, such as CT-1, etc.) is up up and induces the enhancement of JAK-STAT3 activity, which leads to the increase of the STAT3 phosphorylation activity to up regulate the expression of SOCS3. SOCS3, in turn, competes with JAK and inhibits its expression, counteracts a part of STAT3 against the heart. The anti apoptosis effect of muscle cells. If miR-19a can inhibit the expression of SOCS3, indirectly improve the activity of JAK-STAT3 and enhance the anti apoptosis effect in IRI. In order to confirm this speculations, this study carries out three aspects of this study: first, to explore the effect of SOCS3 on the apoptosis of ischemia reperfusion injury and to establish cardiomyocytes in vitro. Hypoxic reoxygenation model, the relationship between SOCS3 and apoptotic protein Bim was confirmed by cell survival test, biochemical test in vitro. Secondly, in vitro, the mechanism of miR-19a on the targeting of SOCS3 was investigated. The expression of protein and the effect of SOCS3 on the apoptosis of myocardial cells were detected by miR-19a overexpression and inhibition experiment. Third, third, In order to investigate the correlation between SOCS-3 and acute myocardial infarction, the patients with acute myocardial infarction were used as the research object to analyze whether the high expression of SOCS3 could predict the major adverse events of the heart. The first part was to explore the relationship between SOCS3 and Bim in the process of myocardial anoxia reoxygenation and the effect on the apoptosis of the cells: the establishment of H9c2 cardiac myocytes. Hypoxia reoxygenation model was used to determine the effect of SOCS3 on Bim expression and apoptosis in hypoxia reoxygenation. Methods: 1) cultured H9c2 myocardial cells.2) simulated ischemia reperfusion injury (Hypoxia reoxygenation, HR) model, Western-blot detection SOCS3 expression.3), designed and synthesized 3 targets for SOCS3 gene Si RNA), Western-blot screened the best SOCS3-si RNA.4) to transfect the best SOCS3-si RNA into H9c2 and give hypoxia reoxygenation. The experiment was divided into 4 groups: blank control group (Control group), HR group, HR+Negative Control group, and 5) detection of cell proliferation activity. CS3, the expression of apoptosis related genes Bim, Bax and Bcl-2 apoptosis protein. Results: 1) SOCS3 expression was obviously up regulated in the process of hypoxia reoxygenation. Compared with the blank control group, the survival rate of the HR group decreased significantly (P0.05), the H9c2 survival rate in the SOCS3-si RNA group was significantly increased (P0.05) and the apoptosis rate decreased significantly (3). Down regulation of expression (P0.01); 4) compared with group HR, SOCS3-si RNA+HR group, Bcl-2 expression was up regulation, Bax expression was down, Bcl-2/Bax ratio increased (P0.05). Conclusion: 1) H9c2 myocardial cells in hypoxia reoxygenation, SOCS3 expression increased obviously; 2) in the hypoxic reoxygenation model of cardiac myocytes, silence SOCS3 can reduce the expression of Bim and expressions; 3) in the myocardial cells in anoxia complex In oxygen model, silent SOCS3 can increase the expression of Bcl-2 4) in the hypoxic reoxygenation model, silent SOCS3 can increase cell viability and reduce cell apoptosis. Second part: the effect of SOCS3 on JAK-STAT3 signaling pathway in anoxic reoxygenated cardiomyocytes and the purpose of miR-19a to regulate it: This study is to establish a HR model for H9c2 cardiomyocytes. The expression of miR-19a in the process of HR, miR-19a mimics or miR-19a inhibitor were transfected in H9c2 to regulate the expression of miR-19a, and the expression of SOCS3, the rate of apoptosis, the expression level of apoptosis related gene protein, and the effect of miR-19a on SOCS3 in the hypoxia reoxygenation of cardiomyocytes and the effect of inducing apoptosis of HR cells were investigated. Method: 1) HR model was established for simulated ischemia reperfusion injury, and miR-19a expression.2 was detected by Q RT-PCR). MiR-19a mimics and miR-19a inhibitor were transfected into H9c2 cells using Lipofectamine3000 respectively. The experiment was divided into 6 groups: blank control group. Tive Control group, HR+miR-19a inhibitor group; 4) CCK8 detection of cell viability, Flow cytometry detection of cell apoptosis rate, Western-blot technology to determine SOCS3, p-STAT3, STAT3, Caspases, Caspases, expressions and expressions. The survival rate of H9c2 cells in group s was significantly increased (P0.05), the rate of apoptosis decreased significantly (P0.05), the survival rate of H9c2 cells in miR-19a inhibitor group decreased significantly (P0.05), the apoptosis rate was significantly increased (P0.05); 3) the overexpression miR-19a obviously inhibited SOCS3, Caspases, protein, inhibition of apoptosis and inhibition of apoptosis The expression of SOCS3, Caspases, Bim and Bax protein was up-regulated, the activity of p-STAT3 was inhibited and the expression of Bcl-2 was down regulated. Conclusion: 1) the expression of miR-19a in H9c2 cardiomyocytes was obviously down regulated after hypoxia reoxygenation; 2) in the process of hypoxia reoxygenation of cardiac myocytes, miR-19a may be mediated by the regulation of apoptosis in SOCS3; the third part of plasma SOCS-3 up regulation and acute myocardium Correlation analysis of poor prognosis Objective: an analysis of the correlation between the up-regulation of human plasma SOCS-3 and the poor prognosis of acute myocardial infarction: 2016.3-2016.10 was selected in the patients with acute myocardial infarction (AMI) in our hospital (n=68), the selection of age and sex in the same period, and the volunteers with no heart blood tube system disease as the healthy control group (n=20). Enzyme linked immunosorbent assay (ELISA) for the determination of SOCS3, C Tn I and CK-MB. and the correlation analysis of SOCS3 and C Tn I, CK-MB, NT-pro, left ventricular ejection fraction, coronary artery disease, cardiac function classification and major adverse cardiovascular events. Compared with the healthy control group (P0.01), the sensitivity and specificity of plasma SOCS-3 in AMI patients were 70 and 85.5 (AUC=0.856).SOCS-3, respectively, with hypertension (P=0.002), left ventricular ejection fraction (P=0.004), e GFR (P0.001), e GFR, and other clinicopathological parameters. In addition, the more coronary stenosis in AMI patients, the higher the plasma SOCS-3 level, the.SOCS-3 level was associated with the Killip cardiac function classification. The higher the level of Killip cardiac function, the higher the plasma SOCS-3 level in patients with.AMI, the higher plasma SOCS-3 than the low plasma SOCS-3 patients had higher major adverse cardiovascular events (MA), according to the median of the binary position (MA). CE) incidence (P0.01). Similarly, high plasma SOCS-3 in patients with lower plasma SOCS-3 has a lower overall survival rate for 6 months (P0.01). Plasma SOCS3 is an independent risk factor for MACE. Conclusion: SOCS-3 is a valuable biological marker for predicting the prognosis of acute myocardial infarction. Myocardial ischemia and reperfusion injury can help us to understand the regulatory mechanism of SOCS3 and further explore the mechanism of myocardial protection.

【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R541

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