雌激素对CVB3诱导的NK细胞表达IFN-γ的调控
发布时间：2018-11-19 12:41
【摘要】：病毒性心肌炎(viral myocarditis,VMC)是临床常见的心血管系统疾病。其中B3型柯萨奇病毒(Coxsackievirus B3,CVB3)在病毒性心肌炎的发病中扮演着重要角色。最新临床报道证实:男性发生心肌纤维炎症损伤的几率显著高于女性,表明病毒性心肌炎有显著的性别差异。然而,对病毒性心肌炎发病的性别差异机制尚不完全清楚。因此,在细胞和分子水平上阐明病毒性心肌炎发病的性别差异的分子机制,具有重要的理论和应用意义。以CVB3感染小鼠建立的心肌炎动物模型,很好的模拟了临床上病毒性心肌炎过程。我们实验室前期的研究结果表明,在病毒性心肌炎小鼠模型中,雄性小鼠的发病程度显著高于雌性小鼠,且雄性小鼠心脏微环境中IFN-γ的表达是雌小鼠的5倍,并且在感染早期主要由NK细胞表达。NK细胞是VMC感染早期最主要的炎性浸润细胞,可直接作用于病毒感染的心肌细胞,并通过分泌IFN-γ等细胞因子,进一步扩大炎性反应。为了验证NK细胞在病毒性心肌炎中的关键作用,我们通过腹腔注射NK细胞中和抗体anti-ASGM1干预小鼠心肌炎模型,发现中和抗体清除NK细胞后雄性小鼠心脏浸润的IFN-γ的表达显著减少,心肌炎性病状减轻。同时我们动态监测到雌雄小鼠感染CVB3后心脏浸润的NK细胞其数量百分比没有显著性别差异,但是雄性小鼠心脏浸润的NK细胞表达IFN-γ的水平要显著高于雌性小鼠。进一步我们给雄性心肌炎小鼠注射雌激素进行干预,结果发现注射雌激素后,雄性小鼠心脏微环境中IFN-γ的表达水平显著下调,提示我们雌激素可能影响了心肌炎小鼠心脏浸润NK细胞IFN-γ的表达。因此,本课题主要探讨病毒性心肌炎发病过程中雌激素如何调控小鼠心脏微环境中NK细胞IFN-γ的表达。为了探究雌激素在病毒性心肌炎中的重要作用,我们首先考虑到自然发育过程中,性发育不成熟的小鼠和性发育成熟雌雄小鼠在感染CVB3后是否有不同的发病状态。我们在构建雄性小鼠半数致死剂量的心肌炎模型过程中发现,性发育不成熟(2周龄)小鼠感染CVB3后没有显著的性别差异,而性发育成熟(6周龄)小鼠感染CVB3后具有显著的性别差异,雄性小鼠的发病程度显著高于雌性小鼠,并且雄性小鼠心脏浸润的NK细胞表达IFN-γ程度显著高于雌性小鼠。我们通过去势手术摘除雌性小鼠的卵巢,发现去势组雌性小鼠心脏炎症明显加重,心脏浸润的NK细胞分泌的IFN-γ较对照组显著上调。以上结果验证了雌激素在调控小鼠心脏浸润的NK细胞分泌IFN-γ过程中的重要作用。我们进一步通过体外实验探究雌激素对NK细胞分泌IFN-γ的调控。我们用雌激素和CVB3处理磁珠分选得到的NK细胞,结果发现,CVB3的刺激可上调NK细胞IFN-γ的分泌,而雌激素可抑制这一现象。并且我们发现,CVB3不能直接感染NK细胞,但是CVB3刺激可上调NK细胞IFN-γ的表达。为了探究CVB3是否直接活化NK细胞,我们在体外模拟了两种感染过程:1)CVB3感染心肌细胞后,与纯化的NK细胞共培养,2)CVB3直接刺激NK细胞,有趣的是,上述实验均能上调NK细胞IFN-γ的表达。已有相关文献报道,雌激素可通过调控活化的淋巴细胞中T-bet的表达来影响IFN-γ的分泌。后续实验我们发现,雌激素的刺激能够在蛋白水平下调CVB3刺激后NK细胞分泌IFN-γ信号通路中T-bet的表达。我们由此猜测:雌激素可通过下调NK细胞T-bet表达来抑制IFN-γ的分泌。基于NK细胞在病毒性心肌炎发病过程中的重要作用,在本研究中我们利用不同性别BALB/c小鼠对CVB3感染后病毒性心肌炎严重程度的差异,着重研究雌激素对NK细胞表达IFN-γ的调控在病毒性心肌炎发病过程中的作用,为阐明病毒性心肌炎发病的性别差异提供新的分子调控机制,同时为病毒性心肌炎的防治提供可能的靶点
[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.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R542.21
[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.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R542.21
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