CAR和DAF在CVB3感染HeLa細(xì)胞中的上調(diào)變化規(guī)律
發(fā)布時(shí)間：2019-06-11 01:57
【摘要】： 目的 柯薩奇病毒(Coxsachievirus,CV)是一類在自然界普遍存在的病毒,屬于小核糖核酸病毒科(Picornaviridas),腸道病毒屬(Enterovirus)�？滤_奇病毒已知有30個(gè)血清型,根據(jù)病毒對(duì)乳鼠的致病特點(diǎn)及對(duì)細(xì)胞敏感性的不同,將病毒分成A組和B組,B組病毒有6個(gè)血清型B1-B6,可引起特征性傳染性胸肋痛,合并腦膜腦炎、心肌炎、肝炎、溶血性貧血等。其中柯薩奇病毒B3型(CVB3)侵入機(jī)體之后,主要導(dǎo)致病毒性心肌炎(viral myocarditis,VMC)、腦膜炎等。從CVB3的感染動(dòng)力學(xué)可得知,CVB3感染的細(xì)胞譜十分廣泛,其感染性及程度的主要因素之一取決于這些細(xì)胞表面柯薩奇病毒受體與病毒的相互作用;通過(guò)分析柯薩奇病毒易感細(xì)胞與非感染細(xì)胞的差異,陸續(xù)發(fā)現(xiàn)了柯薩奇病毒-腺病毒受體(CAR)、衰變加速因子(DAF)等受體。CAR幾乎表達(dá)在所有體細(xì)胞的表面,可能與細(xì)胞的粘接和增殖有關(guān),但確切的生物學(xué)功能至今還不清楚,近期發(fā)現(xiàn)CAR可作為柯薩奇病毒和腺病毒的共同病毒受體,才受到研究者的廣泛關(guān)注。DAF也廣泛表達(dá)于體內(nèi)的多種細(xì)胞,早期發(fā)現(xiàn)DAF可作為一種補(bǔ)體調(diào)節(jié)蛋白,其調(diào)節(jié)補(bǔ)體的功能現(xiàn)已明確,但它作為一種病毒受體的功能卻研究甚少。目前,CVB3具體的致病機(jī)制還不清楚,CVB3結(jié)合受體時(shí),動(dòng)態(tài)構(gòu)象中結(jié)合位點(diǎn)的定位、胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)、細(xì)胞骨架的變化等均值得深入研究。 在本實(shí)驗(yàn)中,用CAR抗體和DAF抗體干預(yù)HeLa細(xì)胞,檢測(cè)在CVB3感染時(shí)CAR抗體和DAF抗體對(duì)細(xì)胞的保護(hù)作用,間接反映出CAR和DAF在CVB3入侵HeLa細(xì)胞過(guò)程中所起作用的大小;并分別在RNA水平和蛋白水平上檢測(cè)CAR和DAF隨病毒感染時(shí)間和濃度的變化規(guī)律。 方法 1、不同濃度的CAR抗體和DAF抗體對(duì)HeLa細(xì)胞的保護(hù)作用: 實(shí)驗(yàn)分為5組:①V(virus)組:即病毒組,加入CVB3與HeLa細(xì)胞相互作用,作為陽(yáng)性對(duì)照;②D(DAF抗體)組:即DAF抗體作用組,在加入CVB3之前,先用DAF抗體與HeLa細(xì)胞相互作用。③C(CAR抗體)組:即CAR抗體作用組,在加入CVB3之前,先用CAR抗體與HeLa細(xì)胞相互作用。④C+D(CAR抗體和DAF抗體)組:即CAR抗體和DAF抗體共同作用組,在加入CVB3之前,先用CAR抗體和DAF抗體共同與HeLa細(xì)胞相互作用。⑤NOR(normal)組:即正常HeLa細(xì)胞對(duì)照組,用正常的HeLa細(xì)胞做為以上各組的陰性對(duì)照。 (1)MTT法檢測(cè)各組細(xì)胞活性:在490nm波長(zhǎng)下檢測(cè)各組細(xì)胞光密度值(OD值),計(jì)算出各組均值、細(xì)胞相對(duì)生存率(RSR)及各組抗體對(duì)病毒的抑制率(IR)。 (2)空斑計(jì)數(shù)法檢測(cè)抗體對(duì)病毒毒力的影響:對(duì)各組的病毒空斑用計(jì)數(shù)器計(jì)數(shù),并計(jì)算出各組的空斑形成單位。 2、反轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)檢測(cè)CARmRNA、DAFmRNA在CVB3入侵HeLa細(xì)胞過(guò)程中的表達(dá)變化:用Trizol提取細(xì)胞總RNA,逆轉(zhuǎn)錄合成cDNA,PCR擴(kuò)增后進(jìn)行瓊脂糖凝膠電泳,紫外燈下觀察電泳結(jié)果并拍照、掃描。將凝膠電泳圖條帶密度進(jìn)行計(jì)算機(jī)分析,計(jì)算電泳條帶與β-actin擴(kuò)增條帶光密度值作為其相對(duì)表達(dá)強(qiáng)度。 3、流式細(xì)胞儀檢測(cè)CAR和DAF在病毒入侵HeLa細(xì)胞過(guò)程中表達(dá)的變化:CVB3與HeLa細(xì)胞相互作用后,加入CAR和DAF的熒光抗體,用流式細(xì)胞儀對(duì)二者的表達(dá)情況進(jìn)行檢測(cè)。 結(jié)果 1、MTT法檢測(cè)結(jié)果:細(xì)胞生存率與抗體濃度呈正相關(guān),在細(xì)胞的生存率方面,D組與V組相比差異無(wú)統(tǒng)計(jì)學(xué)意義,C組與V組相比差異有統(tǒng)計(jì)學(xué)意義(P＜0.05),C+D組與V組相比差異有顯著統(tǒng)計(jì)學(xué)意義(P＜0.01)。在對(duì)病毒的抑制率上,C+D組對(duì)病毒的抑制效果最好,且在抑制效果上C組+D組＜C+D組。 2、空斑計(jì)數(shù)法檢測(cè)結(jié)果:由于抗體的封閉作用,CVB3病毒的毒力有所下降,致病性減弱。在病毒空斑減少程度上以C+D組最為顯著。 3、RT-PCR和流式細(xì)胞儀檢測(cè)結(jié)果顯示:在CVB3作用于HeLa細(xì)胞后,CAR和DAF的表達(dá)出現(xiàn)上調(diào),上調(diào)水平與正常對(duì)照組相比有顯著統(tǒng)計(jì)學(xué)意義(P＜0.01)。1h之后,DAF的表達(dá)恢復(fù)正常,CAR仍維持在較高水平。并且CAR和DAF的上調(diào)幅度與CVB3的濃度成正相關(guān)。 結(jié)論 在CVB3侵入HeLa細(xì)胞過(guò)程中,作為病毒受體的CAR與DAF相比起到更為主要的作用,且CAR和DAF可能具有一定的協(xié)同作用。CVB3的侵入會(huì)上調(diào)DAF和CAR在細(xì)胞表面的表達(dá),且上調(diào)程度與CVB3的濃度呈正相關(guān)。CVB3在侵入細(xì)胞后,細(xì)胞表面DAF的表達(dá)迅速下調(diào),這可能與病毒逃避免疫監(jiān)視有關(guān)。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R373
本文編號(hào)：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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R373
【引證文獻(xiàn)】
相關(guān)博士學(xué)位論文 前1條
1 韓鐵鎖;SSM-CVB3感染獼猴模型的建立及其致病性的研究[D];吉林大學(xué);2012年
，本文編號(hào)：2496913
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