Cx43对休克大鼠血管渗漏和血管低反应性的调节作用及机制

发布时间：2018-04-29 18:02

本文选题：Connexin43(Cx43) + 严重脓毒症　；参考：《第三军医大学》2015年博士论文

【摘要】：休克为战创伤最常见的并发症,其发生率和死亡率都很高。严重创伤/休克病人广泛存在血管功能下降,主要包括两个方面:一是休克后血管反应性下降,即是全身血管对内源性和外源性血管舒缩物质反应性降低或是不反应。这种血管的低反应性严重影响休克的发生发展及治疗,是导致休克晚期血压难以回升和致死的主要原因之一。二是休克后血管屏障功能下降,即血管渗漏,也叫血管通透性升高,特别是创伤失血休克继发的严重脓毒症、脓毒性休克。血管渗漏可使血管内液体外渗,导致组织水肿和有效循环量的进一步下降,影响组织和器官功能,并最终发展为多器官功能障碍(MODS),其死亡率高达40-70%。目前研究证实血管低反应性的发生机制主要有:血管平滑肌细胞膜受体失敏机制、膜超极化及我们实验室提出的钙失敏学说。血管渗漏的机制主要包括两种途径:跨内皮细胞途径和内皮细胞旁途径。这两种途径在休克血管渗漏中发挥着主要作用,血管内皮生长因子(VEGF),凝血酶(thrombin),TNF-α等多种细胞因子主要也是通过这两条通路来影响血管的屏障功能。缝隙连接是一种细胞间的重要结构,在血管中呈现高表达,可介导细胞间多种电化学信号的传导,参与多种血管功能的调控。Cx43是细胞间缝隙连接的主要构成蛋白,研究发现Cx43在多种血管疾病中发挥重要的作用,如:动脉粥样硬化,高血压等。我们前期研究发现Cx43参与休克后血管反应性的调控,但其机制不清。最近有研究显示在酸吸入所导致的急性肺损伤中,Cx43的表达在肺血管的通透性变化中发挥重要作用,那么缝隙连接蛋白Cx43是否会参与在休克后血管渗漏的调节,机制如何?目前尚不清楚。研究已证实Rho激酶信号通路和PKC信号通路在调节细胞骨架、引起细胞收缩和细胞迁移等方面发挥关键的作用。我们前期的研究证实Rho激酶通路和PKC信号通路在休克后血管低反应性的调节中发挥重要的作用。另外也有研究发现在血管渗漏方面,Rho激酶通路也起着关键的作用,如Rho A/Rock通路介导了凝血酶引起的血管内皮通透性的增加。研究证实骨桥蛋白可作为一种信号分子参与与血管相关的多种病理生理变化的调节:如细胞粘附,血管生成和细胞迁移。例如OPN通过改变内皮屏障功能介导了由VEGF诱导的细胞迁移。此外,OPN可激活FAK和ERK信号通路,而这些信号通路可导致细胞的刚度变化和细胞骨架重排。这些生理和病理事件都提示OPN可能与血管内皮屏障功能有密切的关系,但具体的机制不清楚。那么Rho激酶通路和OPN是否参与休克后Cx43对血管渗漏和血管反应性的调节呢,尚不清楚。据此,我们首先利用严重脓毒症大鼠模型和体外LPS刺激肺静脉血管内皮细胞,研究了Cx43在严重脓毒症血管渗漏中的作用及其与Rho激酶,PKC和OPN的关系;第二,利用大鼠出血性休克模型,以及缺血缺氧处理的血管环和血管平滑肌细胞研究了Cx43在休克血管反应性调节中的作用及其与Rho激酶和PKC的关系。研究内容与方法:第一部分Cx43在调节严重脓毒症大鼠血管渗漏中的作用及机制。1.初步探讨Cx43在严重脓毒症血管渗漏中的作用:利用严重脓毒症大鼠及LPS刺激的肺静脉血管内皮细胞(VEC),观察血管渗漏情况和血管Cx43蛋白表达变化;利用慢病毒转染肺静脉血管内皮细胞,改变Cx43的表达,测定了单层VECs的TER和对荧光白蛋白(FITC-BSA)透过率的变化。2.Rho激酶在Cx43调节血管渗漏中的作用及机制研究:利用Cx43高表达和正常的肺静脉血管内皮细胞,观察Rho激酶抑制剂对Cx43调节休克血管渗漏的影响及Cx43对Rho激酶表达的影响。3.OPN/紧密连接蛋白在Cx43调节休克血管渗漏中的作用及机制研究:利用严重脓毒症大鼠和LPS刺激的肺静脉血管内皮细胞,测定OPN,zo-1,claudin-5表达变化及血管渗漏的变化,及OPN的RNA干扰对血管渗漏及zo-1和claudin-5表达的影响;及利用Cx43转染的肺静脉血管内皮细胞,观察Cx43对OPN表达的影响及Cx43对OPN上游转录因子表达的影响。第二部分Cx43在调节失血性休克大鼠血管低反应性中的作用及机制。1.Cx43在PDGF调节血管反应性中的作用及与PKC和Rho激酶的关系:利用失血性休克大鼠及缺氧血管环,观察MEGJ阻断剂和Cx43AODN对休克血管钙敏感性和反应性的影响;利用血管平滑肌细胞,观察了Rho激酶和PKC的抑制剂在PDGF调节血管反应性中的作用,及Cx43AODN在PDGF调节Rho激酶和PKC活性中的作用。2.Cx43在BK调节血管反应性中的作用及机制:利用失血性休克大鼠缺氧处理的血管环,观察MEGJ阻断剂18α-GA对血管反应性的影响,及Rho激酶和PKC抑制剂在Cx43介导BK调节休克血管反应性中的作用及机制。实验结果第一部分Cx43对严重脓毒症血管渗漏的调节作用及机制(一)Cx43在严重脓毒症血管渗漏中的作用Cx43参与了严重脓毒症血管血管渗漏的发生,Cx43表达变化与通透性变化呈正相关,改变Cx43的表达可显著调节血管渗漏。提示Cx43在严重脓毒症血管渗漏中发挥重要的作用。(二)Rho激酶-MLC20在Cx43调节血管渗漏中的作用及机制研究LPS刺激和Cx43高表达可显著升高白蛋白的透过率和降低TER值,改变内皮细胞应激纤维的形态,使细胞呈现向中心收缩。Rho激酶抑制剂Y-27632可减轻透过率的升高和TER值的降低及抑制细胞向心收缩。LPS刺激和Cx43高表达可显著性升高Rho激酶的表达,Cx43RNAi可降低了Rho激酶的表达。LPS刺激和Cx43高表达可显著升高的MLC20的磷酸化水平,Rho激酶抑制剂可抑制MLC20磷酸化升高。Cx43的高表达可上调Rho激酶蛋白表达水平。提示Rho激酶-MLC20通路参与了Cx43对休克血管渗漏的调节(三)OPN/紧密连接蛋白在Cx43调节血管渗漏中的作用及机制研究严重脓毒症大鼠和LPS刺激内皮细胞后,OPN的表达显著升高,zo-1和claudin-5的表达逐渐降低。Cx43高表达显著性地降低了zo-1和claudin-5的表达。Cx43干扰对zo-1和claudin-5的表达没影响,但能阻止LPS刺激引起的zo-1和claudin-5表达的下降。OPN干扰可抑制Cx43对zo-1和claudin-5表达的下调。提示,OPN介导了Cx43对休克血管渗漏的调节。Cx43高表达显著性地升高了Tcf-4和β-catenin的m RNA水平及上调了OPN的表达,β-catenin和Tcf-4的RNA干扰可抑制Cx43对OPN表达的上调。证实了Cx43对OPN的调节是通过转录因子Tcf-4/β-catenin来实现的。第二部分Cx43调节失血性休克大鼠血管反应性及其机制(一)Cx43介导PDGF调节血管反应性及与PKC和Rho激酶的关系PDGF可明显改善休克后血管的反应性,MEGJ的阻断剂18-GA和Cx43AODN抑制了PDGF对休克血管反应性的改善作用。PKC抑制剂和Rho激酶抑制剂可显著抑制PDGF对休克血管反应性和钙敏感性的的改善作用。PDGF可明显升高缺氧血管中Rho激酶底物MYPT和PKC底物Peptag C1的磷酸化,Cx43AODN明显抑制了PDGF对缺氧血管MYPT和Peptag C1的磷酸化改善作用。提示Cx43通过激活PKC和Rho激酶通路,介导了PDGF对休克血管反应性的调节。(二)Cx43在BK调节血管反应性中的作用及机制BK显著改善休克后血管的反应性,MEGJ阻断剂18-GA和Cx43AODN显著性的降低了血管对BK的反应性。BK可磷酸化Cx43的ser368位点及激活Rho激酶,PKC-α和PKC-ε的活性,BK对休克血管的改善作用可被Rho激酶抑制剂和PKC抑制剂所阻断。PKC和Rho激酶的激动剂可改善休克血管对BK的反应性,这种作用可被Cx43AODN所阻断。提示,Cx43通过其ser368位点磷酸化和PKC,Rho激酶通路,参与了BK对休克血管反应性的调节。结论1.Cx43参与了休克后血管通透性的调节,一方面Cx43通过激活Rock1-MLC20通路,使细胞骨架发生改变,细胞出现向心收缩,增大内皮细胞间的缝隙;另一方面,Cx43通过转录因子Tcf-4/β-catenin通路,上调OPN的表达。OPN表达的上调会抑制紧密连接蛋白zo-1和claudin-5的表达,使细胞间紧密连接减少,增大通透性。2.Cx43参与了PDGF和BK对休克血管反应性的调节,通过磷酸化Cx43的ser368位点,调节Rho激酶和PKC的活性,改善休克血管的钙敏感性和反应性。
[Abstract]:Shock is the most common complication of war trauma, its incidence and mortality are very high. Severe trauma / shock patients have extensive vascular function decline, mainly including two aspects: one is the decrease of vascular reactivity after shock, that is, the systemic vascular reactivity to endogenous and exogenous vasomotor substances is reduced or not reacted. Low reactivity seriously affects the development and treatment of shock, which is one of the main causes of the difficult to recover and death of the blood pressure in the late shock. Two is the decrease of blood vessel barrier function after shock, that is, vascular leakage, also called blood vessel permeability, especially the severe sepsis secondary to traumatic hemorrhagic shock, septic shock. Blood vessel leakage can cause blood to cause blood. Fluid extravasation in the tube leads to a further decline in tissue edema and effective circulation, affecting the function of tissue and organs and eventually developing multiple organ dysfunction (MODS). The mortality rate is up to 40-70%.. The mechanism of vascular smooth muscle cell membrane receptor desensitization, membrane hyperpolarization and us The mechanism of calcium desensitization proposed by the laboratory. The mechanism of vascular leakage mainly consists of two pathways: the cross endothelial cell pathway and the endothelial cell side pathway. These two pathways play a major role in shock vascular leakage, and the vascular endothelial growth factor (VEGF), thrombin (thrombin), TNF- alpha and many other cytokines are mainly through these two pathways To affect the barrier function of blood vessels, gap junction is an important structure of intercellular, high expression in the blood vessels, mediating the transmission of multiple electrochemical signals between cells, and participating in the regulation of various vascular functions,.Cx43 is the main constituent of intercellular gap junction, and Cx43 plays an important role in a variety of vascular diseases. For example, atherosclerosis, hypertension, and so on. We have found that Cx43 is involved in the regulation of vascular reactivity after shock, but its mechanism is not clear. Recent studies have shown that in acute lung injury caused by acid inhalation, the expression of Cx43 plays a vital role in the permeability changes of the pulmonary vessels, and whether the gap connexin Cx43 will be involved in the acute lung injury. How is the regulation of vascular leakage after shock? It is not clear now. It has been confirmed that the Rho kinase signaling pathway and the PKC signaling pathway play a key role in regulating the cytoskeleton, causing cell contraction and cell migration. Our previous study confirmed that the Rho kinase pathway and the PKC signaling pathway were in the modulation of vascular hypo response after shock. The Rho kinase pathway also plays a key role in vascular leakage, such as the Rho A/Rock pathway that mediates the increase in vascular endothelial permeability caused by thrombin. The study confirms that osteopontin can be used as a signal molecule to regulate a variety of vascular related pathophysiological changes. Such as cell adhesion, angiogenesis and cell migration. For example, OPN mediated cell migration induced by VEGF by changing the endothelial barrier function. In addition, OPN activates the FAK and ERK signaling pathways, which can lead to cell stiffness changes and cytoskeleton rearrangement. These biological and pathological events suggest that OPN may be associated with vascular endothelial screen. There is a close relationship between the barrier function, but the specific mechanisms are not clear. Then, it is not clear whether the Rho kinase pathway and OPN are involved in the regulation of vascular leakage and vascular reactivity after shock. Accordingly, we first used the rat model of severe sepsis and the external LPS stimulation of the pulmonary venous blood tube endothelial cells to study the blood of Cx43 in severe sepsis. The role of tube leakage and its relationship with Rho kinase, PKC and OPN; second. Using rat hemorrhagic shock model, vascular rings and vascular smooth muscle cells treated by ischemia and anoxia, the role of Cx43 in the regulation of shock vascular reactivity and the relationship with Rho kinase and PKC are studied. The contents and methods of the study are: the first part of Cx43 is regulated strictly. Role and mechanism of vascular leakage in rats with severe sepsis.1. preliminary study of the role of Cx43 in vascular leakage of severe sepsis: the use of severe sepsis rats and LPS stimulated pulmonary venous endothelial cells (VEC) to observe vascular leakage and changes in the expression of vascular Cx43 protein; transfection of the pulmonary vein endothelial cells by lentivirus The expression of Cx43, the TER of single layer VECs and the changes in the transmittance of fluorescent albumin (FITC-BSA), the role and mechanism of.2.Rho kinase in Cx43 regulation of vascular leakage: the effect of Cx43 high expression and normal pulmonary vein endothelial cells, the effect of Rho kinase inhibitor on the regulation of the leakage of shock vessels by Cx43 and Cx43 to Rho kinase Effect and mechanism of.3.OPN/ close connexin in Cx43 regulation of shock vascular leakage: the use of severe sepsis rats and LPS stimulated pulmonary venous endothelial cells to determine the changes of OPN, ZO-1, claudin-5 expression and vascular leakage, and the effect of RNA interference on vascular leakage and expression of ZO-1 and claudin-5 in OPN; and The effects of Cx43 on the expression of OPN and the effect of Cx43 on the expression of transcription factors in the upstream of OPN were observed by Cx43 transfected vascular endothelial cells. Second the role of Cx43 in regulating vascular hyporesponsiveness in rats with hemorrhagic shock and the mechanism of.1.Cx43 in regulating vascular reactivity by PDGF and the relationship with PKC and Rho kinase The effects of MEGJ blockers and Cx43AODN on the calcium sensitivity and reactivity of shock vessels were observed and the effects of Rho kinase and PKC inhibitors on PDGF regulation of vascular reactivity were observed with vascular smooth muscle cells, and the role of Cx43AODN in PDGF regulation of Rho kinase and PKC activity was observed in BK regulating blood. The role and mechanism of tube responsiveness: the effect of MEGJ blocker 18 alpha -GA on vascular reactivity and the role and mechanism of Rho kinase and PKC inhibitor in regulating the reactivity of shock vessels by Cx43 mediated BK in hemorrhagic shock rats, and the regulation of Cx43 on vascular leakage of severe sepsis in the first part of the experiment Role and mechanism (1) the role of Cx43 in severe sepsis vascular leakage Cx43 participates in the occurrence of vascular leakage in severe sepsis. The change of Cx43 expression is positively correlated with the change of permeability, and the expression of Cx43 can significantly regulate vascular leakage. It suggests that Cx43 plays an important role in the leakage of severe sepsis blood tube. (two) Rho kinase -MLC 20 the role and mechanism of Cx43 in regulating vascular leakage, LPS stimulation and high expression of Cx43 can significantly increase the transmittance of albumin and reduce the value of TER, change the morphology of stress fibers in endothelial cells, and make the cells present a central contraction of.Rho kinase inhibitor Y-27632 to reduce the rise of the transmittance and decrease the TER value and inhibit the centripetal contraction of the cells. .LPS stimulation and high expression of Cx43 can significantly increase the expression of Rho kinase, and Cx43RNAi can reduce the phosphorylation level of Rho kinase expression.LPS stimulation and Cx43 high expression of MLC20. Rho kinase inhibitors can inhibit the high expression of.Cx43 phosphorylation and up regulation of the expression of Rho kinase protein. The effect of Cx43 on shock vascular leakage (three) the role and mechanism of OPN/ tight connexin in Cx43 regulation of vascular leakage, the expression of OPN was significantly increased after severe sepsis rats and LPS stimulation of endothelial cells. The expression of ZO-1 and claudin-5 gradually reduced the high expression of.Cx43 and reduced the.Cx43 expression of ZO-1 and claudin-5. Interference has no effect on the expression of ZO-1 and claudin-5, but the decrease of.OPN interference from the expression of ZO-1 and claudin-5 induced by LPS stimulation inhibits the downregulation of ZO-1 and claudin-5 expression by Cx43. It suggests that OPN mediated Cx43 on shock vascular leakage. The expression of RNA in beta -catenin and Tcf-4 inhibits the up regulation of Cx43 on OPN expression. It is confirmed that the regulation of Cx43 to OPN is realized through the transcription factor Tcf-4/ beta -catenin. Second part Cx43 regulates the vascular reactivity and its mechanism in hemorrhagic shock rats. Significantly improved vascular reactivity after shock, MEGJ blockers 18-GA and Cx43AODN inhibit the improvement of PDGF to shock vascular reactivity,.PKC inhibitors and Rho kinase inhibitors can significantly inhibit the improvement of PDGF on shock vascular reactivity and calcium sensitivity..PDGF can significantly increase the MYPT and PKC of Rho kinase substrate in anoxic blood vessels The phosphorylation of substrate Peptag C1, Cx43AODN significantly inhibits the effect of PDGF on the phosphorylation of MYPT and Peptag C1 in anoxic blood vessels. It is suggested that Cx43 is mediated by the activation of PKC and Rho kinase pathway, which mediates the regulation of PDGF on the reactivity of the shock vessels. (two) the role of Cx43 in regulating vascular reactivity and the mechanism of angiogenesis significantly improve the reactivity of blood vessels after shock. MEGJ blockers, 18-GA and Cx43AODN, significantly reduced the ser368 site of the vascular reactive.BK phosphorylated Cx43 and the activation of the Rho kinase, PKC- alpha and PKC- epsilon, and the BK on the shock of the shock vessels could be modified by the inhibitors and inhibitors of the Rho kinase and the activator of the kinase to improve the response to the shock vessel. This effect can be blocked by Cx43AODN. It is suggested that Cx43 participates in the regulation of BK on the vascular reactivity of shock through its phosphorylation of ser368 site and PKC, Rho kinase pathway. Conclusion 1.Cx43 is involved in the regulation of vascular permeability after shock. On the one hand, Cx43 changes the cytoskeleton by activating the Rock1-MLC20 pathway, and the cell appears centripetal contraction. On the other hand, the up regulation of Cx43 through the transcription factor Tcf-4/ beta -catenin pathway up-regulation the expression of.OPN expression of OPN inhibits the expression of tight connexin ZO-1 and claudin-5, reduces the close connection between cells and increases the permeability.2.Cx43 involved in the regulation of PDGF and BK on the reactivity of shock vessels and through phosphorylation Cx4 3 of the ser368 locus regulates the activity of Rho kinase and PKC, and improves calcium sensitivity and responsiveness of shock vessels.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R605.971

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