【摘要】：研究背景：新生小鼠在心尖切除后21天發(fā)生完全的心臟再生,該過(guò)程包括心尖部形成血凝塊包裹心尖切口處、明顯的炎癥反應(yīng)激活、心肌細(xì)胞增殖增加等,其中炎癥反應(yīng)激活是心臟損傷后最先發(fā)生的反應(yīng)之一。既往關(guān)于成年哺乳動(dòng)物的研究顯示炎癥反應(yīng)促進(jìn)纖維疤痕的形成,抑制心臟再生過(guò)程。新生小鼠心臟損傷激發(fā)的炎癥反應(yīng)伴隨早期再生過(guò)程,并未影響其心臟再生能力。急性炎癥反應(yīng)在新生小鼠心臟再生中發(fā)揮怎樣的作用尚未闡明。本研究旨在以新生小鼠心尖切除模型為切入點(diǎn),探討急性炎癥在新生小鼠心臟再生中作用與機(jī)制,進(jìn)而為心臟再生調(diào)控機(jī)制提供新的依據(jù)與線索。研究結(jié)果：一、急性炎癥反應(yīng)在新生小鼠心臟再生中發(fā)揮了重要作用。1)新生1天小鼠心尖切除后,Ly-6G(1A8)陽(yáng)性的炎癥性白細(xì)胞浸潤(rùn)損傷心肌組織,且炎癥反應(yīng)標(biāo)志物(il6, il1b, ccl3)表達(dá)明顯上調(diào),證明新生小鼠心尖切除后發(fā)生了急性炎癥反應(yīng)。2)為了研究單純急性炎癥反應(yīng)在心臟再生中的作用,我們采用當(dāng)今世界上管徑最小的注射針顯微注射酵母多糖A至新生1天小鼠心尖心肌中,構(gòu)建無(wú)菌炎癥模型；通過(guò)Ki67、pH3分別與α-actinin雙染和Brdu追蹤實(shí)驗(yàn)證明單純急性炎癥反應(yīng)可誘導(dǎo)心肌細(xì)胞增殖和心肌細(xì)胞生成。3)為了探索急性炎癥反應(yīng)在新生1天小鼠心臟再生中扮演怎樣的角色,我們?cè)谛募馇谐蠼o予廣譜抗炎藥地塞米松抑制急性炎癥反應(yīng),發(fā)現(xiàn)抑制急性炎癥可以降低心肌細(xì)胞增殖和生成。HE和Masson染色結(jié)果顯示心尖切除后抑制急性炎癥導(dǎo)致心臟不能再生,心臟超聲檢查發(fā)現(xiàn)心尖切除后抑制急性炎癥使心臟心功能明顯受損。二、IL-6/STAT3信號(hào)通路是啟動(dòng)新生小鼠心臟再生的關(guān)鍵機(jī)制。1)通過(guò)細(xì)胞因子芯片和PCR實(shí)驗(yàn)發(fā)現(xiàn)細(xì)胞因子IL-6在新生小鼠心尖切除后表達(dá)上調(diào)最明顯,我們推測(cè)IL-6在新生1天小鼠心臟再生中可能發(fā)揮著重要作用。我們顯微注射IL-6至新生小鼠心尖心肌內(nèi),發(fā)現(xiàn)心肌細(xì)胞增殖和新生成的心肌細(xì)胞均明顯增加。敲除IL-6的新生小鼠心尖切除后顯示心肌細(xì)胞增殖和新生成的心肌細(xì)胞明顯降低,且IL-6-/-小鼠心臟心尖切除21天后心臟不能完全再生,心臟超聲評(píng)價(jià)發(fā)現(xiàn)敲除IL-6的小鼠心尖切除后心功能明顯受損。2)為了確認(rèn)IL-6促進(jìn)新生小鼠心尖切除后心肌細(xì)胞增殖的分子機(jī)制,采用Western實(shí)驗(yàn)篩選IL-6下游3個(gè)信號(hào)通路,分別為STAT3、AKT、Erk1/2通路,發(fā)現(xiàn)STAT3信號(hào)在心尖切除后明顯激活。后續(xù)實(shí)驗(yàn)證明,新生小鼠心肌細(xì)胞中特異性敲除STAT3后使得心尖切除后心肌細(xì)胞增殖明顯降低,且新生成的心肌細(xì)胞明顯減少。結(jié)論：新生小鼠心臟損傷后急性炎癥反應(yīng)可以誘導(dǎo)心肌細(xì)胞增殖來(lái)促進(jìn)心臟再生過(guò)程。IL-6作為促炎因子,通過(guò)下游STAT3信號(hào)通路在啟動(dòng)心臟再生過(guò)程中發(fā)揮了關(guān)鍵作用。
[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 偽 -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.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R541.6

【共引文獻(xiàn)】

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