CAR和DAF在CVB3感染HeLa细胞中的上调变化规律
发布时间：2019-06-11 01:57
【摘要】： 目的 柯萨奇病毒(Coxsachievirus,CV)是一类在自然界普遍存在的病毒,属于小核糖核酸病毒科(Picornaviridas),肠道病毒属(Enterovirus)。柯萨奇病毒已知有30个血清型,根据病毒对乳鼠的致病特点及对细胞敏感性的不同,将病毒分成A组和B组,B组病毒有6个血清型B1-B6,可引起特征性传染性胸肋痛,合并脑膜脑炎、心肌炎、肝炎、溶血性贫血等。其中柯萨奇病毒B3型(CVB3)侵入机体之后,主要导致病毒性心肌炎(viral myocarditis,VMC)、脑膜炎等。从CVB3的感染动力学可得知,CVB3感染的细胞谱十分广泛,其感染性及程度的主要因素之一取决于这些细胞表面柯萨奇病毒受体与病毒的相互作用;通过分析柯萨奇病毒易感细胞与非感染细胞的差异,陆续发现了柯萨奇病毒-腺病毒受体(CAR)、衰变加速因子(DAF)等受体。CAR几乎表达在所有体细胞的表面,可能与细胞的粘接和增殖有关,但确切的生物学功能至今还不清楚,近期发现CAR可作为柯萨奇病毒和腺病毒的共同病毒受体,才受到研究者的广泛关注。DAF也广泛表达于体内的多种细胞,早期发现DAF可作为一种补体调节蛋白,其调节补体的功能现已明确,但它作为一种病毒受体的功能却研究甚少。目前,CVB3具体的致病机制还不清楚,CVB3结合受体时,动态构象中结合位点的定位、胞内信号转导、细胞骨架的变化等均值得深入研究。 在本实验中,用CAR抗体和DAF抗体干预HeLa细胞,检测在CVB3感染时CAR抗体和DAF抗体对细胞的保护作用,间接反映出CAR和DAF在CVB3入侵HeLa细胞过程中所起作用的大小;并分别在RNA水平和蛋白水平上检测CAR和DAF随病毒感染时间和浓度的变化规律。 方法 1、不同浓度的CAR抗体和DAF抗体对HeLa细胞的保护作用: 实验分为5组:①V(virus)组:即病毒组,加入CVB3与HeLa细胞相互作用,作为阳性对照;②D(DAF抗体)组:即DAF抗体作用组,在加入CVB3之前,先用DAF抗体与HeLa细胞相互作用。③C(CAR抗体)组:即CAR抗体作用组,在加入CVB3之前,先用CAR抗体与HeLa细胞相互作用。④C+D(CAR抗体和DAF抗体)组:即CAR抗体和DAF抗体共同作用组,在加入CVB3之前,先用CAR抗体和DAF抗体共同与HeLa细胞相互作用。⑤NOR(normal)组:即正常HeLa细胞对照组,用正常的HeLa细胞做为以上各组的阴性对照。 (1)MTT法检测各组细胞活性:在490nm波长下检测各组细胞光密度值(OD值),计算出各组均值、细胞相对生存率(RSR)及各组抗体对病毒的抑制率(IR)。 (2)空斑计数法检测抗体对病毒毒力的影响:对各组的病毒空斑用计数器计数,并计算出各组的空斑形成单位。 2、反转录聚合酶链式反应(RT-PCR)检测CARmRNA、DAFmRNA在CVB3入侵HeLa细胞过程中的表达变化:用Trizol提取细胞总RNA,逆转录合成cDNA,PCR扩增后进行琼脂糖凝胶电泳,紫外灯下观察电泳结果并拍照、扫描。将凝胶电泳图条带密度进行计算机分析,计算电泳条带与β-actin扩增条带光密度值作为其相对表达强度。 3、流式细胞仪检测CAR和DAF在病毒入侵HeLa细胞过程中表达的变化:CVB3与HeLa细胞相互作用后,加入CAR和DAF的荧光抗体,用流式细胞仪对二者的表达情况进行检测。 结果 1、MTT法检测结果:细胞生存率与抗体浓度呈正相关,在细胞的生存率方面,D组与V组相比差异无统计学意义,C组与V组相比差异有统计学意义(P＜0.05),C+D组与V组相比差异有显著统计学意义(P＜0.01)。在对病毒的抑制率上,C+D组对病毒的抑制效果最好,且在抑制效果上C组+D组＜C+D组。 2、空斑计数法检测结果:由于抗体的封闭作用,CVB3病毒的毒力有所下降,致病性减弱。在病毒空斑减少程度上以C+D组最为显著。 3、RT-PCR和流式细胞仪检测结果显示:在CVB3作用于HeLa细胞后,CAR和DAF的表达出现上调,上调水平与正常对照组相比有显著统计学意义(P＜0.01)。1h之后,DAF的表达恢复正常,CAR仍维持在较高水平。并且CAR和DAF的上调幅度与CVB3的浓度成正相关。 结论 在CVB3侵入HeLa细胞过程中,作为病毒受体的CAR与DAF相比起到更为主要的作用,且CAR和DAF可能具有一定的协同作用。CVB3的侵入会上调DAF和CAR在细胞表面的表达,且上调程度与CVB3的浓度呈正相关。CVB3在侵入细胞后,细胞表面DAF的表达迅速下调,这可能与病毒逃避免疫监视有关。
[Abstract]:Purpose Coxsackie virus (CV) is a kind of virus that is common in nature, belonging to the family of Picornaviridas and Enterovirus. Us). The coxsackie virus is known to have 30 serotypes, and the virus is divided into groups A and B according to the pathogenic characteristics of the virus to the milk and the sensitivity to the cells. The B group has six serotypes B1-B6, which can cause the characteristic infectious chest and rib pain, and the meningoencephalitis and the myocarditis are combined. Hepatitis, Hemolytic anemia and the like, wherein the coxsackie virus B3 type (CVB3) enters the body, and mainly leads to viral myocarditis (VMC), and the brain The infection dynamics of CVB3 can be known, and the cell spectrum of CVB3 infection is very extensive. One of the main factors of the infectivity and extent of CVB3 depends on the interaction of the coxsackie virus receptor and the virus on the surface of these cells; and by analyzing the coxsackie virus susceptible cells and the non-infected cells The coxsackie virus-adenovirus receptor (CAR) and the decay accelerating factor (DAF) were found in succession. The receptor. CAR is almost expressed on the surface of all the somatic cells, which may be related to the adhesion and proliferation of the cells, but the exact biological function is not clear to date. Recently, it has been found that CAR can be used as a common viral receptor of coxsackie virus and adenovirus. Extensive attention. DAF is also widely expressed in various cells in the body. Early detection of DAF can be used as a complement-regulating protein, and its function of regulating complement is now clear, but it is a function of a viral receptor. At present, the specific pathogenesis of CVB3 is not clear, and when CVB3 binds to the receptor, the localization of binding site in the dynamic conformation, the intracellular signal transduction, the changes of the cytoskeleton, etc. are all worth deep. In this experiment, the protective effect of CAR and DAF on the cells was detected by CAR and DAF antibodies, which indirectly reflected the protective effect of CAR and DAF on the cells of HeLa cells. the size of the effect, and the detection of CAR and DAF on the RNA level and the protein level with the time of the virus infection and the time of the virus infection, concentration . Method 1, CAR antibodies at different concentrations and DA The protective effect of F-antibody on HeLa cells: The experiment was divided into five groups: the virus group: the virus group, the addition of CVB3 to HeLa cells as the positive control, and the D (DAF antibody) group: That is, the DAF antibody action group, prior to the addition of CVB3, , with the DAF antibody to interact with the HeLa cells. The CDC (CAR antibody) group: the CAR antibody action group, prior to the addition of CVB3, in that case of a combination of CAR antibody and DAF antibody, the CAR antibody and the DAF antibody are used to act together, and before the addition of the CVB3, the CAR antibody is first And the DAF antibody interacts with the HeLa cell. The cell activity of each group was detected by MTT method: the optical density values (OD values) of each group were detected at the wavelength of 490 nm, and the mean values of each group were calculated and the cells were relative to each other. The inhibition rate (IR) of the antibody against the virus (RSR) and the inhibition rate (IR) of each group of the antibodies against the virus. (2) The effect of the antibody on the virulence of the virus was detected by the plaque count method: for the The expression of CARmRNA and DAFmRNA in HeLa cells was detected by reverse transcription polymerase chain reaction (RT-PCR). The total RNA of cells was extracted with Trizol, and the cDNA was synthesized by reverse transcription. carrying out agarose gel electrophoresis after CR amplification, observing the electrophoresis result under an ultraviolet lamp and taking pictures and scanning, carrying out computer analysis on the band density of the gel electrophoresis graph, The optical density values of the bands of the electrophoresis bands and the antigen-actin were calculated as their relative expression intensity.3. The changes of the expression of CAR and DAF in HeLa cells were detected by flow cytometry: the interaction of CVB3 with HeLa cells. after that, The results showed that the survival rate of the cells was positively correlated with the concentration of the antibody, and the survival of the cells was determined by flow cytometry. In terms of rate, there was no statistical difference between group D and group V, and the difference between group C and group V was poor. The difference of C + D group and group V was significant (P <0.01). And the inhibition effect of the C + D group on the virus is the best, and the C group + D group is less than C + D in the inhibition effect. Group.2. Test results of plaque count method: as a result of The results of RT-PCR and flow cytometry showed that the expression of CAR and DAF was up-regulated and up-regulated after the effect of CVB3 on HeLa cells. There was a significant difference in the control group (P <0.01). after that, The expression of DAF was normal and the CAR was still at a higher level. The up-regulation of CAR and DAF was positively correlated with the concentration of CVB3. In the process of invasion of HeLa cells, CAR, as a viral receptor, plays a more important role in comparison with DAF, and CAR And DAF may have a certain synergistic effect. The invasion of CVB3 may increase the expression of DAF and CAR on the surface of the cell.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R373
本文编号：2496913
[Abstract]:Purpose Coxsackie virus (CV) is a kind of virus that is common in nature, belonging to the family of Picornaviridas and Enterovirus. Us). The coxsackie virus is known to have 30 serotypes, and the virus is divided into groups A and B according to the pathogenic characteristics of the virus to the milk and the sensitivity to the cells. The B group has six serotypes B1-B6, which can cause the characteristic infectious chest and rib pain, and the meningoencephalitis and the myocarditis are combined. Hepatitis, Hemolytic anemia and the like, wherein the coxsackie virus B3 type (CVB3) enters the body, and mainly leads to viral myocarditis (VMC), and the brain The infection dynamics of CVB3 can be known, and the cell spectrum of CVB3 infection is very extensive. One of the main factors of the infectivity and extent of CVB3 depends on the interaction of the coxsackie virus receptor and the virus on the surface of these cells; and by analyzing the coxsackie virus susceptible cells and the non-infected cells The coxsackie virus-adenovirus receptor (CAR) and the decay accelerating factor (DAF) were found in succession. The receptor. CAR is almost expressed on the surface of all the somatic cells, which may be related to the adhesion and proliferation of the cells, but the exact biological function is not clear to date. Recently, it has been found that CAR can be used as a common viral receptor of coxsackie virus and adenovirus. Extensive attention. DAF is also widely expressed in various cells in the body. Early detection of DAF can be used as a complement-regulating protein, and its function of regulating complement is now clear, but it is a function of a viral receptor. At present, the specific pathogenesis of CVB3 is not clear, and when CVB3 binds to the receptor, the localization of binding site in the dynamic conformation, the intracellular signal transduction, the changes of the cytoskeleton, etc. are all worth deep. In this experiment, the protective effect of CAR and DAF on the cells was detected by CAR and DAF antibodies, which indirectly reflected the protective effect of CAR and DAF on the cells of HeLa cells. the size of the effect, and the detection of CAR and DAF on the RNA level and the protein level with the time of the virus infection and the time of the virus infection, concentration . Method 1, CAR antibodies at different concentrations and DA The protective effect of F-antibody on HeLa cells: The experiment was divided into five groups: the virus group: the virus group, the addition of CVB3 to HeLa cells as the positive control, and the D (DAF antibody) group: That is, the DAF antibody action group, prior to the addition of CVB3, , with the DAF antibody to interact with the HeLa cells. The CDC (CAR antibody) group: the CAR antibody action group, prior to the addition of CVB3, in that case of a combination of CAR antibody and DAF antibody, the CAR antibody and the DAF antibody are used to act together, and before the addition of the CVB3, the CAR antibody is first And the DAF antibody interacts with the HeLa cell. The cell activity of each group was detected by MTT method: the optical density values (OD values) of each group were detected at the wavelength of 490 nm, and the mean values of each group were calculated and the cells were relative to each other. The inhibition rate (IR) of the antibody against the virus (RSR) and the inhibition rate (IR) of each group of the antibodies against the virus. (2) The effect of the antibody on the virulence of the virus was detected by the plaque count method: for the The expression of CARmRNA and DAFmRNA in HeLa cells was detected by reverse transcription polymerase chain reaction (RT-PCR). The total RNA of cells was extracted with Trizol, and the cDNA was synthesized by reverse transcription. carrying out agarose gel electrophoresis after CR amplification, observing the electrophoresis result under an ultraviolet lamp and taking pictures and scanning, carrying out computer analysis on the band density of the gel electrophoresis graph, The optical density values of the bands of the electrophoresis bands and the antigen-actin were calculated as their relative expression intensity.3. The changes of the expression of CAR and DAF in HeLa cells were detected by flow cytometry: the interaction of CVB3 with HeLa cells. after that, The results showed that the survival rate of the cells was positively correlated with the concentration of the antibody, and the survival of the cells was determined by flow cytometry. In terms of rate, there was no statistical difference between group D and group V, and the difference between group C and group V was poor. The difference of C + D group and group V was significant (P <0.01). And the inhibition effect of the C + D group on the virus is the best, and the C group + D group is less than C + D in the inhibition effect. Group.2. Test results of plaque count method: as a result of The results of RT-PCR and flow cytometry showed that the expression of CAR and DAF was up-regulated and up-regulated after the effect of CVB3 on HeLa cells. There was a significant difference in the control group (P <0.01). after that, The expression of DAF was normal and the CAR was still at a higher level. The up-regulation of CAR and DAF was positively correlated with the concentration of CVB3. In the process of invasion of HeLa cells, CAR, as a viral receptor, plays a more important role in comparison with DAF, and CAR And DAF may have a certain synergistic effect. The invasion of CVB3 may increase the expression of DAF and CAR on the surface of the cell.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R373
【引证文献】
相关博士学位论文 前1条
1 韩铁锁;SSM-CVB3感染猕猴模型的建立及其致病性的研究[D];吉林大学;2012年
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