白介素28A对柯萨奇病毒B3诱导的病毒性心肌炎小鼠的保护作用

发布时间：2018-06-27 22:14

本文选题：柯萨奇病毒B3 + 心肌炎　；参考：《武汉大学》2015年博士论文

【摘要】：背景：心肌炎是指由于各种原因引起的心肌的炎性病变。通过血清学分析、聚合酶链反应、免疫组织化学、原位杂交和电镜等检测已证实多种病毒可导致病毒性心肌炎,其中又以柯萨奇B组病毒(coxsackie virus B,CVB)最为常见,常可并发扩张性心肌病、左心室功能不全和慢性心力衰竭。使用CVB3感染小鼠能较好模拟人类病毒性心肌炎的病理过程。尽管经过几十年的努力研究,CVB3诱导的病毒性心肌炎的病理机制尚未完全明确。虽然CVB3可通过诱导细胞凋亡、关闭细胞的RNA和蛋白合成或病毒的蛋白酶分解心肌收缩蛋白等机制引起细胞功能不全,强烈的Thl反应也可造成心肌损伤,通过免疫调节或抑制可改善心肌损伤。通常认为病毒的直接损伤、宿主的天然免疫和适应性免疫反应决定急性到亚急性病毒性心肌炎的严重程度,病毒基因组对人类心脏功能和结局目前仍有争议。Ⅲ型干扰素家族包括IFN-λ1(IL-29). IFN-λ 2(IL-28A)和IFN-λ3(IL-28B),与典型的Ⅰ型干扰素类似,在许多病毒感染情况下,Ⅲ型干扰素对病毒有抑制作用,并且具有免疫调节作用,不断有研究发现Ⅲ型干扰素通过诱导IFN效应蛋白如STAT等发挥抗病毒作用。然而,最近有研究发现一些病毒可抑制Ⅲ型干扰素的抗病毒活性。本研究研究IL-28A是否对CVB3诱导的病毒性心肌炎小鼠有保护作用,并通过测定CVB3感染的小鼠心肌组织中信号转导和转录激活因子STAT1和STAT2.凋亡相关蛋白Bcl-2、BAX和Caspase-3的表达,探讨其可能的作用机制。实验方法：四周龄雄性BALB/c系小鼠240只,体重10-12克,随机分组。其中160只为感染小鼠,分为A、B、C、D组,依次分别为感染组(n=40)、低剂量IL-28A治疗组(n=40)、中剂量IL-28A治疗组(n=40)和高剂量IL-28A治疗组(n=40)。通过小鼠腹腔内接种105 PFU(空斑形成单位)CVB3病毒感染小鼠,病毒接种当天定位0天,接种病毒后1小时,分别腹腔内使用100ulPBS(磷酸盐缓冲液)、10ug/kg、20ug/kg和40ug/kgIL-28A(IL-28A均溶于100OulPBS),连续注射4天。80只未感染小鼠,随机分为E组和F组,依次分别为对照组(n=40)和IL-28A对照组(n=40),E组小鼠腹腔内注射100ul PBS,F组小鼠腹腔内注射40 ug/kgIL-28A,连续注射4天。在感染病毒后第4天和第7天,每组分别取10只,麻醉后处死,暴露心脏,取10mg组织做斑块试验,一部分心肌组织冻存备用,剩下的组织以福尔马林固定后做病理学等检查,每组余下20只观察健康状况和生存分析。HE染色观察心肌组织中炎性细胞浸润程度,将心肌炎的病变程度分为0-4级,计算炎症评分；以空斑实验法测定心肌组织中的病毒滴度；以Western blot(免疫印迹法)检测心肌组织中STAT1和STAT2的表达；TUNEL法检测心肌细胞凋亡情况,免疫组织化学法检测凋亡相关蛋白Bcl、2.BAX和Caspase-3的表达。实验结果：第一部分：IL-28A对CVB3诱导的病毒性心肌炎小鼠生存率、心肌病理学评分和病毒滴度的影响1.感染病毒后14天,B组、C组、D组小鼠生存率均为100%,较A组小鼠生存率(40%)明显提高(P0.001)。2.感染病毒后第7天,B组、C组和D组小鼠心肌组织病理学评分依次分别为1.58±0.18、0.93±0.15、0.4±0.1,较A组小鼠心肌组织病理学评分(2.81±0.22)均明显降低(P0.01),D组较A组降低最为显著,并且呈剂量依赖性(P0.01)。3.IL-28A可呈剂量依赖性地降低小鼠心肌组织内病毒滴度,感染病毒后第4天,B组、C组、D组小鼠心肌组织中的病毒滴度依次分别为2257±162PFU.1511± 116PFU.996±107PFU,均明显低于A组6432±291PFU(P0.01),而在病毒感染后第7天,小鼠心肌组织中病毒更为明显地被IL-28A抑制,B组、C组、D组小鼠心肌组织中的病毒滴度依次分别为110±16PFU.70±12PFU.20±5PFU,均明显低于A组550±50PFU(P0.01)。第二部分IL-28A对小鼠心肌组织内STAT1和STAT2蛋白表达的影响W_eztern blot检测结果表明,D组小鼠心肌组织中信号转导和转录激活因子STAT1和STAT2蛋白表达水平均显著高于A组。第三部分IL-28A对心肌组织细胞凋亡的影响1.TUNEL检测结果表明,A组小鼠心肌组织的凋亡指数高于E组(凋亡指数,0.7%±0.05% vs 0.1%±0.02%,P0.01),而D组小鼠心肌组织凋亡指数较A组明显下降(0.39%±0.03%vs0.7%±0.05%,P0.01)。2.免疫组织化学法检测表明,与A组相比,D组小鼠心肌组织凋亡相关蛋白Bcl-2表达增加,BAX和Caspase-3的表达降低,Bcl-2/BAX比值升高。结论本实验得出以下结论：(1)IL-28A对CVB3病毒性心肌炎小鼠有保护作用,提高小鼠生存率,减轻心肌组织炎症和坏死,降低病毒滴度；(2)IL-28A降低CVB3病毒性心肌炎小鼠心肌组织细胞调亡,改变凋亡相关蛋白Bcl-2、BAX和Caspase-3的表达；(3)IL-28A对CVB3病毒性心肌炎小鼠的保护作用可能是通过激活STAT1和STAT2。上述结果表明,IL-28A对CVB3病毒性心肌炎小鼠有保护作用,是潜在的治疗药物。
[Abstract]:Background: myocarditis is an inflammatory disease of the myocardium caused by various causes. By serological analysis, polymerase chain reaction, immunohistochemistry, in situ hybridization and electron microscopy, a variety of viruses have been confirmed to cause viral myocarditis, and the Coxsackie B group virus (Coxsackie virus B, CVB) is the most common and often complicated with dilatation Sexual cardiomyopathy, left ventricular dysfunction, and chronic heart failure. The use of CVB3 infected mice can better simulate the pathological process of human viral myocarditis. Despite decades of hard work, the pathological mechanism of viral myocarditis induced by CVB3 has not been completely clear. Although CVB3 can induce apoptosis and close the RNA and egg of cells The mechanisms of white synthesis or viral protease decomposition of myocardial contractile proteins cause cell dysfunction, and a strong Thl response can also cause myocardial damage, and myocardial damage can be improved by immunoregulation or inhibition. It is generally considered that the direct damage of the virus, the natural and adaptive immune responses of the host determine acute to subacute viral myocardium. The viral genome is still controversial for the human heart function and outcome. Type III interferon family includes IFN- lambda 1 (IL-29), IFN- lambda 2 (IL-28A) and IFN- lambda 3 (IL-28B), similar to typical interferon type I. In many cases of virus infection, type III interferon has an inhibitory effect on the virus and has an immunoregulation effect. It has been found that type III interferon plays an antiviral role by inducing IFN effect proteins such as STAT. However, recent studies have found that some viruses can inhibit the antiviral activity of type III interferon. This study studied whether IL-28A has protective effect on CVB3 induced viral myocarditis in mice and the heart of mice infected by CVB3. The expression of signal transduction and transcriptional activator STAT1 and STAT2. apoptosis related protein Bcl-2, BAX and Caspase-3 were expressed in muscle tissue. The possible mechanism of action was discussed. Experimental methods: 240 male BALB/c mice with a four week age, weight 10-12 grams, randomly grouped. 160 of them were infected mice, divided into A, B, C, D group, respectively, respectively, the infection group (n=40), respectively, low Dose IL-28A treatment group (n=40), medium dose IL-28A treatment group (n=40) and high dose IL-28A treatment group (n=40). Inoculated mice with 105 PFU (plaque forming unit) CVB3 virus, the virus was inoculated for 0 days, and the virus was inoculated for 1 hours. 100ulPBS (phosphate buffer solution), 10ug/kg, 20ug/kg and 40ug/kgIL- were used in the abdominal cavity respectively. 28A (IL-28A all dissolved in 100OulPBS), 4 days of continuous injection of.80 only uninfected mice, randomly divided into E group and F group, respectively, the control group (n=40) and IL-28A control group (n=40), E group mice intraperitoneal injection 100ul PBS, abdominal intraperitoneal injection of 40, continuous injection of 4 days. Fourth days and seventh days after infection of the virus, each group of 10, anesthesia, respectively, each group of 10, anesthesia, anesthesia, 10 anesthesia respectively. After drunken death, the heart was exposed and 10mg tissue was taken for plaque test. A part of the myocardial tissue was frozen and reserved. The remaining tissues were examined by formalin after fixation. The remaining 20 observed the health status and survival analysis by.HE staining to observe the degree of inflammatory cell infiltration in the myocardium, and the degree of myocarditis was divided into 0-4 levels. Calculation of inflammation score; detection of viral titer in myocardial tissue by plaque assay; the expression of STAT1 and STAT2 in myocardial tissue by Western blot (immunoblotting); TUNEL method to detect the apoptosis of myocardial cells, immunohistochemical method to detect the expression of apoptosis related protein Bcl, 2.BAX and Caspase-3. Experimental results: the first part: I The survival rate, myocardial pathology score and virus titer of CVB3 induced viral myocarditis in mice were affected 1. days after 1. infection, the survival rate of group B, C group and D group was 100%, and the survival rate of A group (40%) was significantly increased (P0.001).2. infection virus seventh days, B group, C group and D group of myocardium histopathology score were respectively respectively The myocardial histopathological score of 1.58 + 0.18,0.93 + 0.15,0.4 + 0.1 was significantly lower than that of the A group (P0.01), and the D group decreased the most significantly compared with the A group, and the dose dependent (P0.01).3.IL-28A could reduce the virus titer in the myocardium of mice in a dose-dependent manner, fourth days after infection, B group, C group, and D group of myocardium tissue. The virus titers in the mice were 2257 + 162PFU.1511 + 116PFU.996 + 107PFU respectively, respectively, which were significantly lower than those in the A group 6432 + 291PFU (P0.01), but in the seventh day after the virus infection, the virus was more obviously suppressed by IL-28A in the myocardium of the mice. The viral titers in the myocardium of the B, C and D mice were respectively 110 + 16PFU.70 + 12PFU.20 +. The effect of second part IL-28A on the expression of STAT1 and STAT2 in the myocardium of mice was significantly lower than that of group A (P0.01). The results of W_eztern blot detection showed that the expression of signal transduction and activator STAT1 and STAT2 protein in myocardium of group D was significantly higher than that of A group. The third part of cardiac muscle cell apoptosis was found. The results of 1.TUNEL detection showed that the apoptosis index of myocardial tissue in A group was higher than that of group E (0.7% + 0.05% vs 0.1% + 0.02%, P0.01), and the apoptosis index of myocardial tissue in D group was significantly lower than that in A group (0.39% + 0.03%vs0.7% + 0.05%, P0.01).2. immunohistochemical staining showed that the apoptosis phase of the myocardium in D group was compared with the A group. The expression of Bcl-2 was increased, the expression of BAX and Caspase-3 decreased and the ratio of Bcl-2/BAX increased. Conclusion: (1) IL-28A has a protective effect on mice with CVB3 viral myocarditis, improving the survival rate of mice, reducing inflammation and necrosis of the myocardium, reducing the titer of the virus, and (2) IL-28A reduces the myocardium of CVB3 viral myocarditis in mice. The expression of apoptosis related proteins Bcl-2, BAX and Caspase-3 was altered, and (3) the protective effect of IL-28A on CVB3 virus myocarditis mice may be that IL-28A has protective effect on CVB3 viral myocarditis in mice by activating STAT1 and STAT2., and it is a potential therapeutic drug.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R542.21

【相似文献】