急性炎症在新生小鼠心脏再生中的作用与机制

发布时间：2018-08-06 09:04

【摘要】：研究背景：新生小鼠在心尖切除后21天发生完全的心脏再生,该过程包括心尖部形成血凝块包裹心尖切口处、明显的炎症反应激活、心肌细胞增殖增加等,其中炎症反应激活是心脏损伤后最先发生的反应之一。既往关于成年哺乳动物的研究显示炎症反应促进纤维疤痕的形成,抑制心脏再生过程。新生小鼠心脏损伤激发的炎症反应伴随早期再生过程,并未影响其心脏再生能力。急性炎症反应在新生小鼠心脏再生中发挥怎样的作用尚未阐明。本研究旨在以新生小鼠心尖切除模型为切入点,探讨急性炎症在新生小鼠心脏再生中作用与机制,进而为心脏再生调控机制提供新的依据与线索。研究结果：一、急性炎症反应在新生小鼠心脏再生中发挥了重要作用。1)新生1天小鼠心尖切除后,Ly-6G(1A8)阳性的炎症性白细胞浸润损伤心肌组织,且炎症反应标志物(il6, il1b, ccl3)表达明显上调,证明新生小鼠心尖切除后发生了急性炎症反应。2)为了研究单纯急性炎症反应在心脏再生中的作用,我们采用当今世界上管径最小的注射针显微注射酵母多糖A至新生1天小鼠心尖心肌中,构建无菌炎症模型；通过Ki67、pH3分别与α-actinin双染和Brdu追踪实验证明单纯急性炎症反应可诱导心肌细胞增殖和心肌细胞生成。3)为了探索急性炎症反应在新生1天小鼠心脏再生中扮演怎样的角色,我们在心尖切除后给予广谱抗炎药地塞米松抑制急性炎症反应,发现抑制急性炎症可以降低心肌细胞增殖和生成。HE和Masson染色结果显示心尖切除后抑制急性炎症导致心脏不能再生,心脏超声检查发现心尖切除后抑制急性炎症使心脏心功能明显受损。二、IL-6/STAT3信号通路是启动新生小鼠心脏再生的关键机制。1)通过细胞因子芯片和PCR实验发现细胞因子IL-6在新生小鼠心尖切除后表达上调最明显,我们推测IL-6在新生1天小鼠心脏再生中可能发挥着重要作用。我们显微注射IL-6至新生小鼠心尖心肌内,发现心肌细胞增殖和新生成的心肌细胞均明显增加。敲除IL-6的新生小鼠心尖切除后显示心肌细胞增殖和新生成的心肌细胞明显降低,且IL-6-/-小鼠心脏心尖切除21天后心脏不能完全再生,心脏超声评价发现敲除IL-6的小鼠心尖切除后心功能明显受损。2)为了确认IL-6促进新生小鼠心尖切除后心肌细胞增殖的分子机制,采用Western实验筛选IL-6下游3个信号通路,分别为STAT3、AKT、Erk1/2通路,发现STAT3信号在心尖切除后明显激活。后续实验证明,新生小鼠心肌细胞中特异性敲除STAT3后使得心尖切除后心肌细胞增殖明显降低,且新生成的心肌细胞明显减少。结论：新生小鼠心脏损伤后急性炎症反应可以诱导心肌细胞增殖来促进心脏再生过程。IL-6作为促炎因子,通过下游STAT3信号通路在启动心脏再生过程中发挥了关键作用。
[Abstract]:Background: complete cardiac regeneration occurs 21 days after apical resection in newborn mice. The process includes the formation of blood clots in the apical region surrounding the apical incision, the activation of inflammatory response and the increased proliferation of cardiomyocytes. The activation of inflammatory reaction is one of the first reactions after heart injury. Previous studies in adult mammals have shown that inflammatory responses promote the formation of fibrous scars and inhibit cardiac regeneration. The inflammatory response induced by heart injury in newborn mice was not affected by early regeneration. The role of acute inflammatory response in cardiac regeneration in newborn mice has not been clarified. The purpose of this study was to explore the role and mechanism of acute inflammation in cardiac regeneration of newborn mice by using apical excision model of newborn mice as a cut-in point, and to provide a new basis and clue for the regulation mechanism of cardiac regeneration. Results: 1. Acute inflammatory reaction played an important role in cardiac regeneration in newborn mice. 1) Myocardial tissue was injured by Ly-6G (1A8) positive inflammatory leukocyte infiltration after apical excision in neonatal mice. The expression of inflammatory response markers (il6, il1b, ccl3) was upregulated, which proved that acute inflammatory response occurred after apical resection in neonatal mice.) in order to study the role of simple acute inflammatory reaction in cardiac regeneration, We used the microinjection needle with the smallest diameter in the world to inject yeast polysaccharide A into the apical myocardium of the newborn mice on the first day to establish an aseptic inflammatory model. In order to explore the role of acute inflammatory response in the cardiac regeneration of newborn mice, Ki67 pH 3 and 伪 -actinin double staining and Brdu tracing experiments showed that acute inflammatory reaction could induce cardiomyocyte proliferation and cardiomyocyte formation. We give a broad-spectrum anti-inflammatory drug dexamethasone after apical resection to inhibit acute inflammation. It was found that inhibition of acute inflammation could reduce cardiomyocyte proliferation and production. The results of HE and Masson staining showed that inhibition of acute inflammation after apical resection resulted in heart failure to regenerate. Echocardiography showed that inhibition of acute inflammation after apical resection significantly impaired cardiac function. The signal pathway of IL-6 / STAT3 is the key mechanism of cardiac regeneration in newborn mice. 1) cytokine IL-6 expression was up-regulated most obviously after apical excision in newborn mice by cytokine chip and PCR experiments. We speculate that IL-6 may play an important role in cardiac regeneration of 1 day newborn mice. We microinjected IL-6 into the apical myocardium of newborn mice and found that the proliferation and new generation of cardiomyocytes were significantly increased. Apical excision of neonatal mice with knockout IL-6 showed that myocardial cell proliferation and newly produced cardiomyocytes were significantly decreased, and IL-6 / -r-mouse heart apical resection could not completely regenerate the heart 21 days after apical excision. In order to confirm the molecular mechanism of IL-6 promoting cardiomyocyte proliferation after apical excision in newborn mice, three downstream signal pathways of IL-6 were screened by Western experiment. It was found that the STAT3 signal was activated after apical resection in the STAT _ 3 AKT _ (1 / 2) Erk _ (1 / 2) pathway. Further experiments showed that specific knockout of STAT3 in neonatal mouse cardiomyocytes significantly decreased the proliferation of cardiac myocytes and significantly reduced the number of newly formed cardiomyocytes after apical excision. Conclusion: the acute inflammatory response after cardiac injury in neonatal mice can induce cardiomyocyte proliferation to promote cardiac regeneration. IL-6 plays a key role in the initiation of cardiac regeneration through downstream STAT3 signaling pathway.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R541.6

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