NCC抗柯萨奇病毒B3的作用及机制的研究

发布时间：2018-03-14 07:35

本文选题：病毒性心肌炎　切入点：NCC　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：病毒性心肌炎(Viral myocarditis,VMC)是一种心肌疾病,一般是被病毒感染而导致且发病率非常高,目前常见发病于儿童和青少年。VMC可以被多种病毒诱发。如粘病毒、肠道病毒和腺病毒等等。其中,肠道病毒中的柯萨奇B3型病毒(Coxsackievirus B3,CVB3)较为常见。CVB3病毒是无包膜的单链RNA病毒,常经消化道或呼吸道进入人体并使之感染。病毒性心肌炎在临床上主要表现为炎性细胞浸润和心肌细胞的坏死,部分患者的发病初期没有明显症状,但随着病情的持续发展,可能出现心律失常、心功能不全、房室阻滞、休克等症状,严重者甚至猝死。孕妇若感染则能够发生非麻痹性脊髓灰质炎性病变,使胎儿在子宫内受到感染并能导致畸形。VMC现已严重威胁着人类的健康,但对于VMC的发病机制,目前还不明确,临床上也缺少特效的治疗药物。因此,阐明VMC发病机制和开发研制新型特异性的抗病毒性心肌炎药物是当前的重要任务。NCC是一种新型核苷类似物,在前期研究中已经被证实在体外细胞模型中具有很好的抗CVB3活性。本课题成功构建病毒性心肌炎体外模型,通过三种不同的给药方式,探究了药对CVB3吸附作用的影响、药对CVB3生物合成的影响以及药对CVB3的直接作用。通过检测NCC对CVB3,进一步探讨NCC对CVB3感染的宿主细胞凋亡情况的影响并通过RT-PCR和Western-Blot进一步研究NCC抗CVB3的作用机制。方法:1.分别采用先加药再加病毒,先加病毒再加药,药物对病毒直接作用三种不同给药方式,通过MTT法检测NCC对CVB3的作用。2.选用CVB3病毒感染对数期生长状态良好的Hela细胞。然后用流式细胞仪来检测细胞线粒体膜电位的变化情况;应用逆转录-聚合酶链反应(RT-PCR)法检测NCC对CVB3 TLR3、TLR7、MDA-5表达水平的影响。通过免疫印迹实验(Western-Blot)检测MAPK蛋白水平的变化。结果:1.成功构建CVB3体外细胞模型。TCID50=10-6.32.三种给药方式结果:NCC TC50为1.27 m M。计算NCC对CVB3的IC50值,先加药后加病毒,最后加入培养基的方法:测得IC50值为20.04 m M,TI为0.06;先加病毒后加药液持续培养的方法:测得IC50值为25.27μM,TI为50.26;先将病毒与药液混合2h后加入细胞,再加入培养基的方法:测得IC50值为223.68μM,TI为5.68。3.线粒体膜电位检测结果:与病毒对照组相比,NCC作用后,线粒体膜电位均明显升高(P0.05)。4.RT-PCR检测结果:与病毒对照组相比,NCC作用于CVB3感染的Hela细胞后,细胞相关因子TLR3、TLR7和MDA-5的表达均明显降低(P0.05)。5.Western-Blot检测结果:NCC作用于CVB3感染Hela细胞后,p44/42 MAPK(Erk1/2)的蛋白表达量明显上调(P0.05)。结论:1.NCC抗CVB3的作用可能是影响CVB3的生物合成以及药物对CVB3具有直接作用。2.NCC抗CVB3的主要机制可能与NCC对CVB3感染导致Hela细胞的凋亡的抑制作用有关,从实验结果看出在基因水平上NCC可以抑制相关因子TLR3、TLR7和MDA-5的表达;而在蛋白水平上可以促进p44/42 MAPK(Erk1/2)的表达。
[Abstract]:Viral myocarditis virus (VMC) is a kind of myocardial disease, which is usually caused by virus infection and has a very high incidence. At present, it is common in children and adolescents. VMC can be induced by many kinds of viruses, such as myxovirus. Among them, Coxsackievirus B3 (Coxsackievirus B3CVB3) is more common. CVB3 virus is a single-stranded RNA virus with no capsule. Viral myocarditis often enters the human body through the digestive tract or respiratory tract and infects it. The clinical manifestations of viral myocarditis are mainly inflammatory cell infiltration and myocardial cell necrosis. Some patients have no obvious symptoms at the initial stage of the disease, but with the development of the disease, Arrhythmia, cardiac insufficiency, atrioventricular block, shock and other symptoms, severe or even sudden death. The fetus is infected in the womb and can cause malformation. VMC is a serious threat to human health, but the pathogenesis of VMC is still unclear, and there is a lack of special therapeutic drugs in clinic. It is an important task to elucidate the pathogenesis of VMC and to develop a novel specific anti-viral myocarditis drug. NCCis is a novel nucleoside analogue. In previous studies, it has been proved that it has good antiviral CVB3 activity in vitro cell model. In this paper, we successfully constructed the model of viral myocarditis in vitro, and explored the effect of the drug on the adsorption of CVB3 by three different ways of administration. The effect of NCC on the biosynthesis of CVB3 and the direct action of NCC on CVB3. The effect of NCC on apoptosis of CVB3 infected host cells and the mechanism of NCC anti-#en8# were studied by means of RT-PCR and Western-Blot. Method 1. Add the drug first and then the virus, Add the virus first and then add the drug. The drug acts directly on the virus in three different ways of administration. The effect of NCC on CVB3 was detected by MTT method. Hela cells were infected with CVB3 virus in logarithmic phase and then the changes of mitochondrial membrane potential were detected by flow cytometry. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the effect of NCC on the expression of CVB3 TLR7MDA-5. Western-Blot assay was used to detect the changes of MAPK protein level. Results: 1. The cell model of CVB3 in vitro was successfully constructed. The IC50 value of NCC to CVB3 was calculated by calculating the IC50 value of NCC to CVB3. The method of adding virus first, then adding virus, finally adding medium: the IC50 value was 20.04 m MNTI was 0.06; the method of continuous culture with virus and drug solution: the IC50 value was 25.27 渭 MN TI was 50.26; the virus was mixed with drug solution for 2 hours and then added into cell. The method of adding culture medium: the IC50 value was 223.68 渭 MN TI 5.68.3. The result of mitochondrial membrane potential detection: compared with the virus control group, the effect of IC50 was higher than that of the control group. The results of RT-PCR analysis showed that compared with the control group, the CVB3 infected Hela cells were treated with NCCs. The expression of TLR3, TLR7 and MDA-5 decreased significantly. 5. Western-Blot analysis showed that the protein expression of p44 / 42 MAPK / Erk1 / 2) in CVB3 infected Hela cells was significantly up-regulated by P0.05. conclusion\\\%\\%\%\%\%\%\%\%\%\%\%\%\%\%\%\%\%\%\%\%\%\%\%\%\%\%\%\%\%\%\%\%\%\%\%\%\%\%? 2. The main mechanism of CVB3 against CVB3 may be related to the inhibitory effect of NCC on the apoptosis of Hela cells induced by CVB3 infection. The results showed that NCC could inhibit the expression of TLR3, TLR7 and MDA-5 at the gene level, while at the protein level, it could promote the expression of p44 / 42 MAPKN Erk 1 / 2.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96

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