【摘要】：病毒性心肌炎(viral myocarditis,VMC)是臨床常見的心血管系統(tǒng)疾病。其中B3型柯薩奇病毒(Coxsackievirus B3,CVB3)在病毒性心肌炎的發(fā)病中扮演著重要角色。最新臨床報道證實:男性發(fā)生心肌纖維炎癥損傷的幾率顯著高于女性,表明病毒性心肌炎有顯著的性別差異。然而,對病毒性心肌炎發(fā)病的性別差異機制尚不完全清楚。因此,在細胞和分子水平上闡明病毒性心肌炎發(fā)病的性別差異的分子機制,具有重要的理論和應用意義。以CVB3感染小鼠建立的心肌炎動物模型,很好的模擬了臨床上病毒性心肌炎過程。我們實驗室前期的研究結(jié)果表明,在病毒性心肌炎小鼠模型中,雄性小鼠的發(fā)病程度顯著高于雌性小鼠,且雄性小鼠心臟微環(huán)境中IFN-γ的表達是雌小鼠的5倍,并且在感染早期主要由NK細胞表達。NK細胞是VMC感染早期最主要的炎性浸潤細胞,可直接作用于病毒感染的心肌細胞,并通過分泌IFN-γ等細胞因子,進一步擴大炎性反應。為了驗證NK細胞在病毒性心肌炎中的關鍵作用,我們通過腹腔注射NK細胞中和抗體anti-ASGM1干預小鼠心肌炎模型,發(fā)現(xiàn)中和抗體清除NK細胞后雄性小鼠心臟浸潤的IFN-γ的表達顯著減少,心肌炎性病狀減輕。同時我們動態(tài)監(jiān)測到雌雄小鼠感染CVB3后心臟浸潤的NK細胞其數(shù)量百分比沒有顯著性別差異,但是雄性小鼠心臟浸潤的NK細胞表達IFN-γ的水平要顯著高于雌性小鼠。進一步我們給雄性心肌炎小鼠注射雌激素進行干預,結(jié)果發(fā)現(xiàn)注射雌激素后,雄性小鼠心臟微環(huán)境中IFN-γ的表達水平顯著下調(diào),提示我們雌激素可能影響了心肌炎小鼠心臟浸潤NK細胞IFN-γ的表達。因此,本課題主要探討病毒性心肌炎發(fā)病過程中雌激素如何調(diào)控小鼠心臟微環(huán)境中NK細胞IFN-γ的表達。為了探究雌激素在病毒性心肌炎中的重要作用,我們首先考慮到自然發(fā)育過程中,性發(fā)育不成熟的小鼠和性發(fā)育成熟雌雄小鼠在感染CVB3后是否有不同的發(fā)病狀態(tài)。我們在構(gòu)建雄性小鼠半數(shù)致死劑量的心肌炎模型過程中發(fā)現(xiàn),性發(fā)育不成熟(2周齡)小鼠感染CVB3后沒有顯著的性別差異,而性發(fā)育成熟(6周齡)小鼠感染CVB3后具有顯著的性別差異,雄性小鼠的發(fā)病程度顯著高于雌性小鼠,并且雄性小鼠心臟浸潤的NK細胞表達IFN-γ程度顯著高于雌性小鼠。我們通過去勢手術摘除雌性小鼠的卵巢,發(fā)現(xiàn)去勢組雌性小鼠心臟炎癥明顯加重,心臟浸潤的NK細胞分泌的IFN-γ較對照組顯著上調(diào)。以上結(jié)果驗證了雌激素在調(diào)控小鼠心臟浸潤的NK細胞分泌IFN-γ過程中的重要作用。我們進一步通過體外實驗探究雌激素對NK細胞分泌IFN-γ的調(diào)控。我們用雌激素和CVB3處理磁珠分選得到的NK細胞,結(jié)果發(fā)現(xiàn),CVB3的刺激可上調(diào)NK細胞IFN-γ的分泌,而雌激素可抑制這一現(xiàn)象。并且我們發(fā)現(xiàn),CVB3不能直接感染NK細胞,但是CVB3刺激可上調(diào)NK細胞IFN-γ的表達。為了探究CVB3是否直接活化NK細胞,我們在體外模擬了兩種感染過程:1)CVB3感染心肌細胞后,與純化的NK細胞共培養(yǎng),2)CVB3直接刺激NK細胞,有趣的是,上述實驗均能上調(diào)NK細胞IFN-γ的表達。已有相關文獻報道,雌激素可通過調(diào)控活化的淋巴細胞中T-bet的表達來影響IFN-γ的分泌。后續(xù)實驗我們發(fā)現(xiàn),雌激素的刺激能夠在蛋白水平下調(diào)CVB3刺激后NK細胞分泌IFN-γ信號通路中T-bet的表達。我們由此猜測:雌激素可通過下調(diào)NK細胞T-bet表達來抑制IFN-γ的分泌�；贜K細胞在病毒性心肌炎發(fā)病過程中的重要作用,在本研究中我們利用不同性別BALB/c小鼠對CVB3感染后病毒性心肌炎嚴重程度的差異,著重研究雌激素對NK細胞表達IFN-γ的調(diào)控在病毒性心肌炎發(fā)病過程中的作用,為闡明病毒性心肌炎發(fā)病的性別差異提供新的分子調(diào)控機制,同時為病毒性心肌炎的防治提供可能的靶點
[Abstract]:Viral myocarditis (VMC) is a common cardiovascular system. The coxsackievirus B3 (CVB3) plays an important role in the pathogenesis of viral myocarditis. The most recent clinical reports confirm that the probability of the inflammatory injury of the heart muscle fiber in the male is significantly higher than that of the female, indicating that the viral myocarditis has a significant gender difference. However, the mechanism of the gender difference in the pathogenesis of viral myocarditis is not completely clear. Therefore, it is of great theoretical and practical significance to elucidate the molecular mechanism of the sex difference in the pathogenesis of viral myocarditis at the cellular and molecular level. The animal model of myocarditis was established by CVB3, and the viral myocarditis was well simulated. The results from the early stage of our laboratory show that in the mouse model of viral myocarditis, the incidence of the male mice is significantly higher than that of the female mice, and the expression of the IFN-1 in the micro-environment of the heart of the male mice is 5 times that of the female mice, and is mainly expressed by the NK cells in the early stage of the infection. NK cells are the most important inflammatory infiltrating cells in the early stage of VMC infection, can act directly on the myocyte of the virus infection, and further expand the inflammatory reaction by secreting the cytokines such as IFN-1 and the like. In order to verify the key role of NK cells in viral myocarditis, we tested the mouse myocarditis model by intraperitoneal injection of NK cells and anti-ASGM1, and found that the expression of IFN-1 in the heart of the male mice after the clearance of NK cells in the neutralizing antibody was significantly reduced, and the venereal disease of the myocarditis was reduced. At the same time, we dynamically monitor that the percentage of NK cells in the heart-infiltrating NK cells after CVB3 infection in male and female mice is not significantly different, but the level of the NK cell expressing IFN-1 in the heart of the male mice is significantly higher than that of the female mice. The results showed that the expression of IFN-1 in the heart of the male mice decreased significantly after the injection of the estrogen, suggesting that the estrogen may have an effect on the expression of IFN-1 in the heart of the mice with myocarditis. Therefore, this topic mainly discusses how the estrogen regulates the expression of the NK cell IFN-1 in the mouse's heart microenvironment during the pathogenesis of viral myocarditis. In order to explore the important role of estrogen in viral myocarditis, we first take into account that in the course of natural development, the immature mouse and the mature male and female mice of the sex development have different disease states after the infection of CVB3. We found that in the process of constructing half lethal dose of the male mice, it was found that there was no significant gender difference in the development of sexual development (2-week-old) in the mice infected with CVB3, and the sexual development of the mice (6-week-old) had significant gender differences after the infection of CVB3. The degree of incidence of male mice was significantly higher than in female mice, and the expression of IFN-in NK cells in the heart of male mice was significantly higher than in female mice. We removed the ovaries of the female mice by castration, and found that the inflammation of the heart in the female mice of the castrated group was significantly increased, and the IFN-1 secreted by the heart-infiltrated NK cells was significantly up-regulated in the control group. The above results verify the important role of the estrogen in the regulation of the release of IFN-1 from the NK cells in the heart of the mouse. We further explore the regulation of the secretion of IFN-1 by estrogen on NK cells by in vitro experiments. The results show that the stimulation of CVB3 can increase the secretion of IFN-1 in NK cells, and the estrogen can inhibit this phenomenon. And we found that CVB3 could not directly infect NK cells, but CVB3 stimulation could increase the expression of NK cell IFN-1. In order to investigate whether CVB3 directly activates NK cells, we have simulated two infection processes in vitro: 1) After CVB3 is infected with cardiomyocytes, co-culture with purified NK cells, 2) CVB3 directly stimulates NK cells, it is interesting that the above-mentioned experiments can increase the expression of NK cell IFN-1. It has been reported that the estrogen can influence the secretion of IFN-antigen by regulating the expression of T-bet in activated lymphocytes. In a follow-up experiment, we found that the stimulation of estrogen can reduce the expression of T-bet in the IFN-linked signaling pathway after the protein level is down-regulated by CVB3. We have thus speculated that the estrogen can inhibit the secretion of IFN-1 by down-regulation of the NK cell T-bet expression. Based on the important role of NK cells in the pathogenesis of viral myocarditis, we used different sex BALB/ c mice to change the severity of viral myocarditis after CVB3 infection. The role of estrogen on the regulation of the expression of IFN-1 in NK cells in the pathogenesis of viral myocarditis was studied, and a new molecular control mechanism was provided to explain the gender differences in the pathogenesis of viral myocarditis, and a possible target for the prevention and treatment of viral myocarditis was provided.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R542.21

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