肠道病毒71型感染人脑微血管内皮细胞的机制研究

发布时间：2018-05-10 18:29

本文选题：肠道病毒71型 + 脑微血管内皮细胞　；参考：《南方医科大学》2012年博士论文

【摘要】：一、研究背景和目的肠道病毒71型(enterovirus71, EV71)属于小核糖核酸病毒科肠道病毒属的成员,归属于人类肠道病毒A。目前已知EV71的感染可以导致手足口病、疱疹性咽峡炎、无菌性脑膜炎、脑炎和脊髓灰质炎样的麻痹性疾病等多种与神经系统相关的疾病,严重病理症状通常在儿童中发生。自1974年Schmidt等人首次报道从美国加利福尼亚暴发的表现为神经系统症状疾病(1969-1973年)的患者中分离到EV71,随后,世界上许多国家相继报道了EV71病毒在不同地区的流行情况,EV71病毒已在世界范围内引起多次暴发与流行。尽管EV71的基因组和生命周期相似于小核糖核酸病毒科的其它成员,但相应引起的病理症状明显不同；至今尚无特异性的治疗方法及有效的针对性的疫苗预防EV71的感染,因此明确EV71的致病机制对控制婴幼儿手足口病的流行与有效治疗该疾病具有十分重要的意义。相关研究证实在EV71患者中枢神经系统(central nervous system, CNS)的脑干、神经元等部位都已检测到EV71基因及抗原的存在,证明EV71能进入CNS。目前,对于EV71进入CNS的途径有两种理论,如同脊髓灰质炎：病毒入血穿血脑屏障或通过末梢神经沿逆轴突运输感染CNS。有关研究证实新生小鼠口服EV71后,早期可引起持续性病毒血症和血脑脊液屏障通透性增加,推测EV71可通过血运途径传播,但脑组织低水平的病毒数量提示血源性途径并非累及CNS的主要途径,所以EV71穿血脑屏障(blood brain barrier, BBB)机制还未明确。众所周知,BBB是循环和CNS之间一道主要的屏障,它限制着不同物质在两个部位之间的自由运输,对维持CNS的体内平衡起着一个关键的角色,故明确EV71穿BBB的机制对于防治EV71所致的CNS感染有重要意义,这亦是我们想探讨的内容。我们知道,病毒感染过程的第一步是病毒通过与位于细胞表面的特异性受体结合而吸附于被感染的细胞的表面,然后利用细胞的内吞作用进入细胞或将病毒的核酸释放进细胞。所以我们推测构成BBB的主要成分之一内皮细胞可能是EV71的易感细胞,EV71与其结合而导致BBB结构改变和功能障碍,从而感染中枢神经系统。本研究采用分离鉴定的来自于重症手足口病患者的EV71毒株感染HBMEC,形态学观察HBMEC的细胞病变、透射电镜检测HBMEC中EV71的超微结构与Real-Time PCR方法以检测HBMEC细胞培养上清液中EV71拷贝数变化情况,以探讨EV71是否能感染HBMEC并能在其中复制；且进一步检测EV71是否可以诱导HBMEC细胞骨架的改变并诱导其凋亡。最后,因为病毒成功实现对宿主细胞的感染并在细胞内复制需要克服细胞对病毒感染产生的各种免疫防卫反应,阻断或影响宿主细胞的正常循环机制,利用宿主细胞的物质和能量合成病毒自身物质,这种相互作用最终会导致宿主细胞蛋白表达模式的改变,这种改变影响着宿主细胞的正常生理功能,决定和反映着病毒感染致病的进程和结果。为了研究EV71感染HBMEC后宿主细胞蛋白表达模式的改变,为揭示病毒与HBMEC的相互作用机制、病毒的分子致病机制、寻找病毒的作用靶标等研究提供有意义的信息。本研究采用2-D技术分析HBMEC感染EV71后1h、16h、24h时间点与正常HBMEC的培养上清和细胞内的蛋白表达差异点,然后对差异蛋白点进行酶切,提取肽片段,最后用MALDI-TOF/TOF-MS质谱技术获得PMF MSMS图,并利用Internet上的蛋白质数据库对肽质量进行检索,寻找具有相似肽质量指纹图的蛋白质,鉴定了差异率2.5的蛋白。并采用免疫印迹方法进一步对其中波形蛋白(vimentin)差异蛋白的变化进行了验证。二、方法 1、分离与鉴定来自于临床诊断为重症手足口病患者的EV71毒株采用来自于临床诊断为重症手足口病患者的粪便或肛拭子,经肠道病毒通用型引物及EV71特异性引物检测初步诊断为EV71感染。再经RD细胞分离增殖病毒,并采用EV71(226bp)引物与EV71VP1全基因序列(1086bp)引物进行RT-PCR再次鉴定,并测序与blast比对证实。 2、EV71对人脑微血管内皮细胞的感染采用已分离鉴定的EV71毒株感染HBMEC并设置空白对照组：观察HBMEC感染EV71后不同时间点的形态改变,并采用兔抗-EV71多克隆抗体检测HBMEC内EV71抗原的存在,透射电镜直接观察EV71作用HBMEC内是否存在EV71病毒颗粒,并采用(226bp)EV71特异性引物对EV71感染HBMEC和RD细胞不同时间点的培养上清液行定量Real-Time PCR,以检测HBMEC和RD细胞感染EV71不同时间点分泌的EV71拷贝数变化以得出EV71在HBMEC的复制趋势图。 3、采用Jc-1线粒体早期凋亡试剂盒检测EV71感染HBMEC8h时能否导致HBMEC的早期线粒体膜电位降低；采用Annexin-V FITC/PI kit检测EV71能否导致HBMEC的凋亡；采用罗丹明-鬼笔环肽染色以探讨EV71感染HBMEC能否导致HBMEC细胞骨架的重排。 4、EV71感染HBMEC差异蛋白质组学的研究采用分离EV71毒株在不同时间点感染HBMEC,收集裂解细胞,采用Bradford染色液定量蛋白,一向等电聚焦,二向聚丙烯酰胺凝胶电泳(SDS-PAGE),并进行银染和考染,银染胶用来分析、考染胶进行质谱鉴定。采用ImageScanner扫描仪对胶进行扫描,凝胶图像用ImageMaster7.0进行分析。设置差异点倍数为2.5倍，对差异点进行确认，，剔除单块胶个别丰度过高或低的点。挖取差异点进行酶切，通过基质辅助激光解析电离飞行时间质谱和蛋白信息数据库建立差异蛋白质谱。并对鉴定出的蛋白进行功能分析,并采用免疫印迹技术进一步验证鉴定出的差异蛋白vimentin在HBMEC感染EV71不同时间点及正常HBMEC中的表达。三、统计分析数据均用均数±标准差表示,采用SPSS13.0进行统计分析。不同时间点的凋亡率以及HBMEC与RD细胞两组间的EV71拷贝数比较采用两个重复测量因素的方差分析,线粒体凋亡采用独立样本t检验分析。P0.05认为有统计学意义,相关统计图采用Excel或Origin绘图软件制作四、结果 1、分离鉴定出一株重症EV71毒株。 2、从形态学观察,EV71可以导致HBMEC的细胞病变,随着感染时间延长,病变程度愈严重；通过透射电镜可以发现EV71处理HBMEC内存在直径20-30nm的病毒颗粒；采用抗EV71多克隆抗体进一步证实EV71组HBMEC内EV71抗原的存在；而采用EV71特异性引物行实时荧光定量PCR表明EV71能感染HBMEC,并且可在HBMEC内复制,其拷贝数在感染第3天达到最高峰,其复制最高峰出现时间比EV71易感细胞RD细胞早,但最终拷贝数低于RD细胞(P0.01)；通过采用罗丹明-鬼笔环肽染色证实EV71感染HBMEC能导致HBMEC细胞骨架的重排；另外采用JC-1试剂盒证实EV71能在8h诱导HBMEC早期凋亡；采用annexin-V FITC/PI凋亡检测试剂盒检测到EV71能诱导HBMEC的凋亡,而随着感染时间的延长,主要以细胞坏死为主,感染时间对EV71感染所诱导的凋亡和坏死有显著影响(P0.01)。 3、通过双向凝胶电泳与质谱鉴定,初步鉴定了HBMEC感染EV71在0、1、16、24h的28个差异表达蛋白。 4、通过免疫印迹对EV71感染HBMEC与HBMEC的差异表达蛋白进一步验证,证实EV71感染HBMEC可以诱导Vimentin的表达下调,与蛋白质组学结果一致。五、结论 1、我们分离鉴定了来自于重症手足口病患者的EV71野生毒株,体外模拟EV71感染HBMEC模型。 2、通过形态学观察及免疫荧光和透射电镜方法证实EV71能感染HBMEC;采用Real-time PCR方法进一步证实EV71能在HBMEC内复制,这说明HBMEC是EV71的易感细胞,但复制效率低于RD细胞。HBMEC上可能存在EV71的特异性受体,EV71与BBB内皮细胞上特异性受体结合进而通过内吞作用进入细胞里面复制,导致其形态与功能改变而穿BBB入脑。 3、采用JC-1线粒体凋亡检测试剂盒证实EV71感染HBMEC能诱导其线粒体膜电位降低,并且采用Annex-V FITC/PI染色方法证实EV71能诱导HBMEC的凋亡,随着感染时间延长,细胞主要以坏死为主。说明EV71可诱导HBMEC线粒体的凋亡,而线粒体结构和功能障碍是各种刺激因素诱导细胞凋亡的中心事件,并由此导致线粒体内凋亡诱发因子的释放,参与对细胞凋亡的调控,而引起了HBMEC的凋亡。 4、EV71感染HBMEC能导致HBMEC细胞微丝结构的重排,而微丝结构的改变可能对于病毒的复制和活化也起到一定的辅助作用。 5、蛋白质组学研究共发现EV71感染HBMEC进程中28个差异表达蛋白,按功能分为9类。结合差异谱中表达量发生改变的蛋白的功能与EV71感染和未感染HBMEC蛋白表达量变化分析,发现了一些在感染中有重要意义的分子靶标。 6、Vimentin差异表达蛋白可能在EV71感染HBMEC事件中起着一定的作用。
[Abstract]:First, research background and purpose
The enterovirus 71 (enterovirus71, EV71) is a member of the genus enterovirus of the family small ribonucleic virus, belonging to human enterovirus A.. The current known infection of EV71 can lead to hand foot and mouth disease, herpetic angina, aseptic meningitis, encephalitis, and poliomyelitis like paralysis diseases, and a variety of nervous system related diseases. Severe pathological symptoms usually occur in children. Since 1974, Schmidt and others first reported that EV71 was isolated from patients with neurological symptoms (1969-1973 years) outbreaks in California, United States. Subsequently, many countries in the world reported the prevalence of EV71 virus in different regions. The EV71 virus has been cited worldwide. Although the genome and life cycle of EV71 are similar to other members of the family of small ribonucleic virus, the corresponding pathological symptoms are obviously different. There are no specific treatment methods and effective targeted vaccines to prevent EV71 infection. Therefore, it is clear that the pathogenesis of EV71 is to control infants and feet. The prevalence of oral diseases is of great importance in the effective treatment of the disease.
Relevant studies have confirmed that the presence of EV71 genes and antigens has been detected in the brain stem and neurons of the central nervous system (central nervous system, CNS) in EV71 patients. It is proved that EV71 can enter CNS. currently. There are two theories for EV71 entering CNS, like spinal gray matter: the virus enters the blood barrier or passes through the end of the nerve. CNS. related studies of the trans axon transport infection confirmed that after the oral EV71 was taken by the newborn mice, the early onset of sustained viremia and the permeability of the blood cerebrospinal fluid barrier (CSF) barrier increased, and it was suggested that EV71 could be transmitted through the blood transport pathway, but the number of low levels of the virus in the brain suggests that the blood source pathway is not the main pathway involved in CNS, so EV71 is wearing the blood brain barrier (blood). The mechanism of brain barrier, BBB) is not yet clear. As we all know, BBB is a major barrier between circulation and CNS. It restricts the free transport of different substances between two parts and plays a key role in maintaining the balance of CNS in the body. Therefore, it is important for the mechanism of EV71 to wear BBB to prevent CNS infection caused by EV71. We know that the first step of the virus infection process is that the virus is adsorbed on the surface of the infected cell by binding to the specific receptor located on the surface of the cell and then using the endocytosis of the cell to enter the cell or release the virus's nucleic acid into the cell. So we speculate that the main components of the BBB are the main components of the virus. An endothelial cell may be a susceptible cell of EV71. EV71 is associated with BBB structural changes and dysfunction and thus infects the central nervous system. In this study, the isolated EV71 strains from patients with severe hand foot and mouth disease infected HBMEC, morphological observation of HBMEC cytopathic lesions, and transmission electron microscopy to detect the ultrastructure of EV71 in HBMEC. Structure and Real-Time PCR method to detect the change of EV71 copy number in the supernatant of HBMEC cell culture, to explore whether EV71 can infect HBMEC and be able to copy it, and further detect whether EV71 can induce the change of HBMEC cytoskeleton and induce its apoptosis. Finally, the virus successfully infects the host cell and is in the cell Internal replication needs to overcome the various immune defense responses of the cell to virus infection, blocking or affecting the normal circulation mechanism of the host cells, using the substance and energy of the host cells to synthesize the virus itself. This interaction ultimately leads to the change in the protein expression pattern of the host cell, which affects the positive cell of the host. Normal physiological functions, determine and reflect the process and results of viral infection. In order to study the changes in the protein expression patterns of host cells after EV71 infection, the interaction mechanism of the virus and HBMEC, the molecular pathogenesis of the virus, and the target for the search for the action of the virus are provided. This study uses the 2-D technology. After HBMEC infection of EV71, 1H, 16h, 24h time point and normal HBMEC culture supernatant and protein expression in cells were different. Then, the difference protein points were cut by enzyme, and peptide fragments were extracted. Finally, the PMF MSMS map was obtained by MALDI-TOF/TOF-MS mass spectrometry, and the peptide quality was retrieved by using the egg white matter database on Internet to find the phase of the peptide. The protein of peptide mass fingerprinting was identified with a difference rate of 2.5, and the difference of vimentin (vimentin) protein was verified by immunoblotting.
Two, method
1, isolation and identification of EV71 strains from clinically diagnosed patients with severe hand foot mouth disease.
The fecal or anal swabs from patients with severe hand foot and mouth disease were preliminarily diagnosed as EV71 infection by common primers of enterovirus and EV71 specific primers, and then RD cells were isolated and proliferating, and EV71 (226bp) primers and EV71VP1 full gene sequence (1086bp) primers were used to reidentify RT-PCR, and sequencing and blas were sequenced and Blas. T alignment proved.
2, EV71 infection of human brain microvascular endothelial cells
The isolated EV71 strains were infected with HBMEC and the blank control group was set up. The morphological changes of HBMEC infected EV71 at different time points were observed and the existence of EV71 antigen in HBMEC was detected by Rabbit anti -EV71 polyclonal antibody. The presence of EV71 virus particles in HBMEC was observed directly by transmission electron microscopy, and (226bp) EV71 specificity was used. The culture supernatant of EV71 infected HBMEC and RD cells at different time points was used for quantitative Real-Time PCR to detect the EV71 copy number of HBMEC and RD cells infected with EV71 at different time points to obtain the replication trend of EV71 in HBMEC.
3, whether the early mitochondrial membrane potential of EV71 infection was detected by Jc-1 mitochondrial early apoptosis kit could lead to the decrease in the early mitochondrial membrane potential of HBMEC; the detection of EV71 could lead to the apoptosis of HBMEC by Annexin-V FITC/PI kit, and the Luo Danming phelic cyclic peptide staining was used to investigate whether EV71 infection HBMEC could lead to the rearrangement of the skeleton of the HBMEC cells.
4, EV71 infection HBMEC differential proteomics
The isolated EV71 strains were infected with HBMEC at different time points, collecting lysate cells, using Bradford staining liquid quantitative protein, always isoelectric focusing, two polyacrylamide gel electrophoresis (SDS-PAGE), silver staining and staining, silver staining gel used for analysis, test gel for qualitative identification. The gel was scanned with ImageScanner scanner and gel was used to scan the gel. The image was analyzed with ImageMaster7.0. The difference point multiplier was 2.5 times, the difference points were confirmed, the individual abundances were excessively high or low. The difference points were removed and the enzyme was cut. The differential protein mass spectrometry was built by the matrix assisted laser analytical ionization time of flight mass spectrometry and protein information database. Functional analysis and immunoblotting were used to further verify the expression of the differential protein vimentin in HBMEC infected EV71 at different time points and in normal HBMEC.
Three, statistical analysis
The data were expressed with mean standard deviation, and SPSS13.0 was used for statistical analysis. The rate of apoptosis at different time points and the number of EV71 copies between the two groups of HBMEC and RD cells were compared with the variance analysis of two repeated measurement factors. The mitochondrial apoptosis was analyzed by independent sample t test and.P0.05 recognition was statistically significant, and the correlation statistical map was Excel Or Origin drawing software
Four, the result
1, a severe EV71 strain was isolated and identified.
2, from morphological observation, EV71 can lead to the cytopathic disease of HBMEC. With the prolonged infection, the more serious the lesion is, EV71 can be found by transmission electron microscopy to deal with the virus particles in 20-30nm in the diameter of HBMEC, and the existence of EV71 antigen in the EV71 group HBMEC is further confirmed by anti EV71 polyclonal antibody; and EV71 specific induction is used. Real time fluorescence quantitative PCR shows that EV71 can infect HBMEC, and can be replicated in HBMEC, and its copy number reaches the peak at third days of infection. The peak time of its replication peak appears earlier than that of EV71 cell RD cells, but the final copy number is lower than that of RD cells (P0.01); and the EV71 infection HBMEC can lead to HBM by using rhodamine phiric cyclic peptide staining. The rearrangement of EC cytoskeleton, and JC-1 kit confirmed that EV71 could induce apoptosis in early HBMEC by 8h, and annexin-V FITC/PI apoptosis detection kit was used to detect the apoptosis of HBMEC, but with the prolongation of the infection time, it was mainly cell necrosis, and the time of infection had a significant effect on the apoptosis and necrosis induced by EV71 infection. Ringing (P0.01).
3, two dimensional gel electrophoresis and mass spectrometry were used to identify 28 differentially expressed proteins of HBMEC infected with EV71 in 0,1,16,24h.
4, the differential expression protein of HBMEC and HBMEC infected by EV71 was further verified by immunoblotting. It was confirmed that EV71 infection HBMEC could induce the downregulation of Vimentin expression, which was consistent with the proteomic results.
Five. Conclusion
1, we isolated and identified EV71 wild strains from severe HFMD patients, and simulated the EV71 infection HBMEC model in vitro.
2, EV71 can infect HBMEC by morphological observation, immunofluorescence and transmission electron microscopy, and Real-time PCR method is used to further confirm that EV71 can be replicated in HBMEC, which indicates that HBMEC is a susceptible cell of EV71, but the replication efficiency is lower than that of EV71 receptor on RD cell.HBMEC. EV71 and specific receptors on endothelial cells are specific. In combination with endocytosis, it enters the cell to replicate, resulting in its morphological and functional changes and wearing BBB into the brain.
3, the JC-1 mitochondrial apoptosis detection kit confirmed that EV71 infection HBMEC could induce the decrease of mitochondrial membrane potential, and the Annex-V FITC/PI staining method proved that EV71 could induce apoptosis of HBMEC. With the prolongation of the infection time, the cells were mainly necrotic. It shows that EV71 can induce the apoptosis of HBMEC mitochondria, and the mitochondrial structure and functional barrier can be induced by EV71. It is the central event of inducing apoptosis by various stimulant factors, and thus leads to the release of apoptosis inducing factors in mitochondria, and participates in the regulation of apoptosis, and causes the apoptosis of HBMEC.
4, EV71 infection with HBMEC can lead to rearrangement of HBMEC cell microfilament structure, and the change of microfilament structure may play a supporting role in virus replication and activation.
5, a total of 28 differentially expressed proteins in the process of EV71 infection were found in the proteomics study, and they were divided into 9 categories according to their function. The function of the protein expressed in the differential spectrum and the changes in the expression of EV71 and uninfected HBMEC protein were analyzed, and some important molecular targets in the infection were found.
6, Vimentin differentially expressed proteins may play a role in EV71 infection of HBMEC.

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R373

【引证文献】