本文選題：柯薩奇病毒B3 + 心肌炎��； 參考：《武漢大學(xué)》2015年博士論文

【摘要】：背景：心肌炎是指由于各種原因引起的心肌的炎性病變。通過血清學(xué)分析、聚合酶鏈反應(yīng)、免疫組織化學(xué)、原位雜交和電鏡等檢測已證實多種病毒可導(dǎo)致病毒性心肌炎,其中又以柯薩奇B組病毒(coxsackie virus B,CVB)最為常見,常可并發(fā)擴(kuò)張性心肌病、左心室功能不全和慢性心力衰竭。使用CVB3感染小鼠能較好模擬人類病毒性心肌炎的病理過程。盡管經(jīng)過幾十年的努力研究,CVB3誘導(dǎo)的病毒性心肌炎的病理機(jī)制尚未完全明確。雖然CVB3可通過誘導(dǎo)細(xì)胞凋亡、關(guān)閉細(xì)胞的RNA和蛋白合成或病毒的蛋白酶分解心肌收縮蛋白等機(jī)制引起細(xì)胞功能不全,強(qiáng)烈的Thl反應(yīng)也可造成心肌損傷,通過免疫調(diào)節(jié)或抑制可改善心肌損傷。通常認(rèn)為病毒的直接損傷、宿主的天然免疫和適應(yīng)性免疫反應(yīng)決定急性到亞急性病毒性心肌炎的嚴(yán)重程度,病毒基因組對人類心臟功能和結(jié)局目前仍有爭議。Ⅲ型干擾素家族包括IFN-λ1(IL-29). IFN-λ 2(IL-28A)和IFN-λ3(IL-28B),與典型的Ⅰ型干擾素類似,在許多病毒感染情況下,Ⅲ型干擾素對病毒有抑制作用,并且具有免疫調(diào)節(jié)作用,不斷有研究發(fā)現(xiàn)Ⅲ型干擾素通過誘導(dǎo)IFN效應(yīng)蛋白如STAT等發(fā)揮抗病毒作用。然而,最近有研究發(fā)現(xiàn)一些病毒可抑制Ⅲ型干擾素的抗病毒活性。本研究研究IL-28A是否對CVB3誘導(dǎo)的病毒性心肌炎小鼠有保護(hù)作用,并通過測定CVB3感染的小鼠心肌組織中信號轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄激活因子STAT1和STAT2.凋亡相關(guān)蛋白Bcl-2、BAX和Caspase-3的表達(dá),探討其可能的作用機(jī)制。實驗方法：四周齡雄性BALB/c系小鼠240只,體重10-12克,隨機(jī)分組。其中160只為感染小鼠,分為A、B、C、D組,依次分別為感染組(n=40)、低劑量IL-28A治療組(n=40)、中劑量IL-28A治療組(n=40)和高劑量IL-28A治療組(n=40)。通過小鼠腹腔內(nèi)接種105 PFU(空斑形成單位)CVB3病毒感染小鼠,病毒接種當(dāng)天定位0天,接種病毒后1小時,分別腹腔內(nèi)使用100ulPBS(磷酸鹽緩沖液)、10ug/kg、20ug/kg和40ug/kgIL-28A(IL-28A均溶于100OulPBS),連續(xù)注射4天。80只未感染小鼠,隨機(jī)分為E組和F組,依次分別為對照組(n=40)和IL-28A對照組(n=40),E組小鼠腹腔內(nèi)注射100ul PBS,F組小鼠腹腔內(nèi)注射40 ug/kgIL-28A,連續(xù)注射4天。在感染病毒后第4天和第7天,每組分別取10只,麻醉后處死,暴露心臟,取10mg組織做斑塊試驗,一部分心肌組織凍存?zhèn)溆?剩下的組織以福爾馬林固定后做病理學(xué)等檢查,每組余下20只觀察健康狀況和生存分析。HE染色觀察心肌組織中炎性細(xì)胞浸潤程度,將心肌炎的病變程度分為0-4級,計算炎癥評分；以空斑實驗法測定心肌組織中的病毒滴度；以Western blot(免疫印跡法)檢測心肌組織中STAT1和STAT2的表達(dá)；TUNEL法檢測心肌細(xì)胞凋亡情況,免疫組織化學(xué)法檢測凋亡相關(guān)蛋白Bcl、2.BAX和Caspase-3的表達(dá)。實驗結(jié)果：第一部分：IL-28A對CVB3誘導(dǎo)的病毒性心肌炎小鼠生存率、心肌病理學(xué)評分和病毒滴度的影響1.感染病毒后14天,B組、C組、D組小鼠生存率均為100%,較A組小鼠生存率(40%)明顯提高(P0.001)。2.感染病毒后第7天,B組、C組和D組小鼠心肌組織病理學(xué)評分依次分別為1.58±0.18、0.93±0.15、0.4±0.1,較A組小鼠心肌組織病理學(xué)評分(2.81±0.22)均明顯降低(P0.01),D組較A組降低最為顯著,并且呈劑量依賴性(P0.01)。3.IL-28A可呈劑量依賴性地降低小鼠心肌組織內(nèi)病毒滴度,感染病毒后第4天,B組、C組、D組小鼠心肌組織中的病毒滴度依次分別為2257±162PFU.1511± 116PFU.996±107PFU,均明顯低于A組6432±291PFU(P0.01),而在病毒感染后第7天,小鼠心肌組織中病毒更為明顯地被IL-28A抑制,B組、C組、D組小鼠心肌組織中的病毒滴度依次分別為110±16PFU.70±12PFU.20±5PFU,均明顯低于A組550±50PFU(P0.01)。第二部分IL-28A對小鼠心肌組織內(nèi)STAT1和STAT2蛋白表達(dá)的影響W_eztern blot檢測結(jié)果表明,D組小鼠心肌組織中信號轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄激活因子STAT1和STAT2蛋白表達(dá)水平均顯著高于A組。第三部分IL-28A對心肌組織細(xì)胞凋亡的影響1.TUNEL檢測結(jié)果表明,A組小鼠心肌組織的凋亡指數(shù)高于E組(凋亡指數(shù),0.7%±0.05% vs 0.1%±0.02%,P0.01),而D組小鼠心肌組織凋亡指數(shù)較A組明顯下降(0.39%±0.03%vs0.7%±0.05%,P0.01)。2.免疫組織化學(xué)法檢測表明,與A組相比,D組小鼠心肌組織凋亡相關(guān)蛋白Bcl-2表達(dá)增加,BAX和Caspase-3的表達(dá)降低,Bcl-2/BAX比值升高。結(jié)論本實驗得出以下結(jié)論：(1)IL-28A對CVB3病毒性心肌炎小鼠有保護(hù)作用,提高小鼠生存率,減輕心肌組織炎癥和壞死,降低病毒滴度；(2)IL-28A降低CVB3病毒性心肌炎小鼠心肌組織細(xì)胞調(diào)亡,改變凋亡相關(guān)蛋白Bcl-2、BAX和Caspase-3的表達(dá)；(3)IL-28A對CVB3病毒性心肌炎小鼠的保護(hù)作用可能是通過激活STAT1和STAT2。上述結(jié)果表明,IL-28A對CVB3病毒性心肌炎小鼠有保護(hù)作用,是潛在的治療藥物。
[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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R542.21

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