肠道病毒71型感染人脑微血管内皮细胞的机制研究
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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。P<0.05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(P<0.01)；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(P<0.01)。
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。
1. Background and objective
EV71belongs to the human enterovirus, a species of the enterovirus genus within the family Picornaviridae. It is a common cause of hand, foot and mouth disease, aseptic meningitis, encephalitis, poliomyelitis-like paralysis and a lot of epidemics correlated with nerve system. EV71associated with neurological diseases was first reported in1974, the documented outbreak in California. After the first identification, the epidemic of EV71in different regions have been reported, it have been lead to repeatedly outbreak and epidemic in the world. Although the genome and the life cycle of EV71is similar to the picornavirus family members, but it induced pathological symptoms are significantly different. The mechanisms of EV71pathogenesis still remain unclear.
There is no specific treatment and effective vaccine to prevent EV71infection. It has very important significance that clearly the pathogenesis of EV71. Studies have also detected EV71viral genomes and antigens by histopathology, immunohistochemistry, and reverse transcription polymerase chain reaction in the cells of CNS tissue, including neurons, neuronal processes and associated inflammatory cells. This indicate EV71can into CNS, however, it is not clearly how EV71infected CNS. As for poliovirus (PV), two possible routes by which the virus reaches the central nervous system (CNS) have been suggested:the virus either enters the CNS from the blood across the blood-brain barrier (BBB) or is transmitted to the CNS through peripheral nerves via retrograde axonal transport. Although the study confirmed after newborn mice taken orally EV71, it may be a cause of persistent viremia and blood cerebrospinal fluid barrier permeability increasely. But low levels of virus number in CNS indicated hematogenous pathway is not main way. Therefore, it has important significance for the prevention and treatment of EV71infected how EV71effects on the structure and function of BBB. The BBB constitutes the interface between the blood and the central nervous system (CNS). Under physiological conditions, the BBB maintains CNS homeostasis and selectively regulates intracellular and paracellular passage of ions, molecules and cells. So it is not clearly how EV71penetrate BBB. Virus infection process is the first step in virus by and located at the cell surface receptor binding and specificity of adsorbed on the surface of infected cells, then the cells by endocytosis into the cell or virus nucleic acids are released into the cell.
Viruses use specific receptor to bind target cells. Then the virus partical by endocytosis into cells and the virus nucleic acids are released into cells. Therefore, we hypothesized the major components of BBB (endothelial cells) might be susceptible cell on EV71. EV71can lead to the structural changes and dysfunction of BBB. Although researchers have reported cynomolgus monkeys and young mice as animal models to represent CNS involved diseases by EV71infection. However, some differences still remain between EV71replication sites in these animal models and those in humans. Therefore, it is still room for find better model to unmask neural pathogenesis of EV71. In this study, EV71was isolated from the stool of a patient with poliomyelitis-like paralytic disease. HBMEC was infected by EV71. To explore whether EV71can infect and replicate in HBMEC, the morphological of HBMEC was observed, EV71in the HBMEC was observed by transmission electron microscopic and amount of viral RNA of EV71in the culture supernatants were detected by real-time PCR method. The cytoskeletal alterations and apoptosis of HBMEC were detected.
Viral invasion will modify the patterns of host cell protein expression, which may affect the normal physiological function of host cell and determines viral pathogenic progress and consequence. Therefore, studies on viral infections proteomics contributes to uncover the mechanism of interaction between EV71and HBMEC and viral molecular pathogenesis, found early biomarker of EV71infection, develop earlier diagnostic method, evaluate therapeutic effect and prognosis and so on. In this paper, techniques of viral infection proteomics, the progress of changes of HBMEC proteome induced by EV71were explored. The protein expression of culture supernatants and intracellular at Oh,1h,16h,24h after EV71infected HBMEC were analyzed by two-dimensional-electrophoresis and matrix assisted laser-desorption ionization time of flight mass spectrometry.
2. Methods
2.1. An EV71strain was isolated and identificated which come from severe hand-foot-mouth disease patients.
2.2. HBMEC was infected by EV71, the CPE were observed; EV71antigen in HBMEC was observed by immunofluorescence; EV71in the HBMEC was observed by transmission electron microscopic and amount of viral RNA of EV71in the culture supernatants was detected by real-time PCR method. The cytoskeletal alterations and apoptosis of HBMEC were detected. EV71related HBEMC and normal HBMEC were examined by2-D. Intensity changes of protein spots detected with statistic significance were identified by MALDI-TOF MS or MS/MS. To evaluate the reliability and accurate of the proteomic results, we used western blot technique to detect expression of vimentin in the development of EV71ralated HBMEC.
3. Statistical analysis
All data in this study are presented as mean±SD. All these statistical analyses were carried out using prism6.0or SPSS13.0for window.The apotosis rate and EV71copies were evaluated by the ANOVA for the repeated measures, The normality of results was evaluated by the Kolmogorov-Smirnov test. Comparison between groups were performed with the independent samples t-test. P<0.05was considered statistically significant.
4. Results
4.1. An EV71strain was isolated.
4.2. According to the morphological observation, EV71can result in CPE of HBEMC, along with the prolonged of infection, the CPE became more severe. Diameter20-30nm virus particles was found in HBEMC by transmission electron microscope. EV71antigen was founded in HBMEC by using a polyclonal antibody. The copy number of EV71in HBMEC and RD cells were detected by using Real-Time PCR method, the copy number reached peak at third days, the replication effect in HBMEC was lower than in RD cells (P<0.01). EV71infection can lead to HBMEC cytoskeletal rearrangement by using rhodamine phalloidin staining. After HBEMC was infected by EV71at8h, the early apoptosis of HBMEC was detected with JC-1kit. Using the annexin V-FITC/PI kit, EV71can lead to the apoptosis of HBMEC, accompany with the prolong of infection time, mainly in cell necrosis (P<0.01).
4.3. By two-dimensional gel electrophoresis and mass spectrometry identification,28proteins were found expressed differently in these groups.
4.3. To evaluate the reliability and accurate of the proteomic results, we used western blot technique to detect expression of vimentin protein and demonstrated the expression of vimentin can lead to cytoskeletal alterations.
5. Conclusion
5.1. The morphological observation and immunofluorescence and transmission electron microscopy method confirmed HBMEC can be infected with EV71.EV71can replicated in HBMEC. This indicated HBMEC is susceptible cell of EV71, however, the replication efficiency is more lower than RD. BBB may exist specific receptors of EV71. EV71may be binding with endothelial cells of BBB and through endocytosis into cells, at last, lead to the morphological and functional changes of BBB and invasion into the brain.
5.2. Using JC-1mitochondrial apoptosis detection kit has confirmed infection of EV71can induce the mitochondrial apoptosis in HBMEC, along with the prolonged duration of infection, cells mainly in necrosis.
5.3. HBMEC cell micro filaments structural rearrangements by EV71infected HBMEC. Microfilament cytoskeleton changes also played a supporting role for viral replication and activation.
5.4. Proteomics research discovered28expression of protein, according to the functions are divided into9categories. Combining differential spectrum expression change of protein function and the infection of EV71attack and attack HBMEC protein expression variation analysis, some important molecular targets were found.
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。P<0.05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(P<0.01)；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(P<0.01)。
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。
1. Background and objective
EV71belongs to the human enterovirus, a species of the enterovirus genus within the family Picornaviridae. It is a common cause of hand, foot and mouth disease, aseptic meningitis, encephalitis, poliomyelitis-like paralysis and a lot of epidemics correlated with nerve system. EV71associated with neurological diseases was first reported in1974, the documented outbreak in California. After the first identification, the epidemic of EV71in different regions have been reported, it have been lead to repeatedly outbreak and epidemic in the world. Although the genome and the life cycle of EV71is similar to the picornavirus family members, but it induced pathological symptoms are significantly different. The mechanisms of EV71pathogenesis still remain unclear.
There is no specific treatment and effective vaccine to prevent EV71infection. It has very important significance that clearly the pathogenesis of EV71. Studies have also detected EV71viral genomes and antigens by histopathology, immunohistochemistry, and reverse transcription polymerase chain reaction in the cells of CNS tissue, including neurons, neuronal processes and associated inflammatory cells. This indicate EV71can into CNS, however, it is not clearly how EV71infected CNS. As for poliovirus (PV), two possible routes by which the virus reaches the central nervous system (CNS) have been suggested:the virus either enters the CNS from the blood across the blood-brain barrier (BBB) or is transmitted to the CNS through peripheral nerves via retrograde axonal transport. Although the study confirmed after newborn mice taken orally EV71, it may be a cause of persistent viremia and blood cerebrospinal fluid barrier permeability increasely. But low levels of virus number in CNS indicated hematogenous pathway is not main way. Therefore, it has important significance for the prevention and treatment of EV71infected how EV71effects on the structure and function of BBB. The BBB constitutes the interface between the blood and the central nervous system (CNS). Under physiological conditions, the BBB maintains CNS homeostasis and selectively regulates intracellular and paracellular passage of ions, molecules and cells. So it is not clearly how EV71penetrate BBB. Virus infection process is the first step in virus by and located at the cell surface receptor binding and specificity of adsorbed on the surface of infected cells, then the cells by endocytosis into the cell or virus nucleic acids are released into the cell.
Viruses use specific receptor to bind target cells. Then the virus partical by endocytosis into cells and the virus nucleic acids are released into cells. Therefore, we hypothesized the major components of BBB (endothelial cells) might be susceptible cell on EV71. EV71can lead to the structural changes and dysfunction of BBB. Although researchers have reported cynomolgus monkeys and young mice as animal models to represent CNS involved diseases by EV71infection. However, some differences still remain between EV71replication sites in these animal models and those in humans. Therefore, it is still room for find better model to unmask neural pathogenesis of EV71. In this study, EV71was isolated from the stool of a patient with poliomyelitis-like paralytic disease. HBMEC was infected by EV71. To explore whether EV71can infect and replicate in HBMEC, the morphological of HBMEC was observed, EV71in the HBMEC was observed by transmission electron microscopic and amount of viral RNA of EV71in the culture supernatants were detected by real-time PCR method. The cytoskeletal alterations and apoptosis of HBMEC were detected.
Viral invasion will modify the patterns of host cell protein expression, which may affect the normal physiological function of host cell and determines viral pathogenic progress and consequence. Therefore, studies on viral infections proteomics contributes to uncover the mechanism of interaction between EV71and HBMEC and viral molecular pathogenesis, found early biomarker of EV71infection, develop earlier diagnostic method, evaluate therapeutic effect and prognosis and so on. In this paper, techniques of viral infection proteomics, the progress of changes of HBMEC proteome induced by EV71were explored. The protein expression of culture supernatants and intracellular at Oh,1h,16h,24h after EV71infected HBMEC were analyzed by two-dimensional-electrophoresis and matrix assisted laser-desorption ionization time of flight mass spectrometry.
2. Methods
2.1. An EV71strain was isolated and identificated which come from severe hand-foot-mouth disease patients.
2.2. HBMEC was infected by EV71, the CPE were observed; EV71antigen in HBMEC was observed by immunofluorescence; EV71in the HBMEC was observed by transmission electron microscopic and amount of viral RNA of EV71in the culture supernatants was detected by real-time PCR method. The cytoskeletal alterations and apoptosis of HBMEC were detected. EV71related HBEMC and normal HBMEC were examined by2-D. Intensity changes of protein spots detected with statistic significance were identified by MALDI-TOF MS or MS/MS. To evaluate the reliability and accurate of the proteomic results, we used western blot technique to detect expression of vimentin in the development of EV71ralated HBMEC.
3. Statistical analysis
All data in this study are presented as mean±SD. All these statistical analyses were carried out using prism6.0or SPSS13.0for window.The apotosis rate and EV71copies were evaluated by the ANOVA for the repeated measures, The normality of results was evaluated by the Kolmogorov-Smirnov test. Comparison between groups were performed with the independent samples t-test. P<0.05was considered statistically significant.
4. Results
4.1. An EV71strain was isolated.
4.2. According to the morphological observation, EV71can result in CPE of HBEMC, along with the prolonged of infection, the CPE became more severe. Diameter20-30nm virus particles was found in HBEMC by transmission electron microscope. EV71antigen was founded in HBMEC by using a polyclonal antibody. The copy number of EV71in HBMEC and RD cells were detected by using Real-Time PCR method, the copy number reached peak at third days, the replication effect in HBMEC was lower than in RD cells (P<0.01). EV71infection can lead to HBMEC cytoskeletal rearrangement by using rhodamine phalloidin staining. After HBEMC was infected by EV71at8h, the early apoptosis of HBMEC was detected with JC-1kit. Using the annexin V-FITC/PI kit, EV71can lead to the apoptosis of HBMEC, accompany with the prolong of infection time, mainly in cell necrosis (P<0.01).
4.3. By two-dimensional gel electrophoresis and mass spectrometry identification,28proteins were found expressed differently in these groups.
4.3. To evaluate the reliability and accurate of the proteomic results, we used western blot technique to detect expression of vimentin protein and demonstrated the expression of vimentin can lead to cytoskeletal alterations.
5. Conclusion
5.1. The morphological observation and immunofluorescence and transmission electron microscopy method confirmed HBMEC can be infected with EV71.EV71can replicated in HBMEC. This indicated HBMEC is susceptible cell of EV71, however, the replication efficiency is more lower than RD. BBB may exist specific receptors of EV71. EV71may be binding with endothelial cells of BBB and through endocytosis into cells, at last, lead to the morphological and functional changes of BBB and invasion into the brain.
5.2. Using JC-1mitochondrial apoptosis detection kit has confirmed infection of EV71can induce the mitochondrial apoptosis in HBMEC, along with the prolonged duration of infection, cells mainly in necrosis.
5.3. HBMEC cell micro filaments structural rearrangements by EV71infected HBMEC. Microfilament cytoskeleton changes also played a supporting role for viral replication and activation.
5.4. Proteomics research discovered28expression of protein, according to the functions are divided into9categories. Combining differential spectrum expression change of protein function and the infection of EV71attack and attack HBMEC protein expression variation analysis, some important molecular targets were found.
引文
[1]王晓华,李文琳.小儿手足口病1602例报告.中华流行病学杂志,2001(04)：37-38.
[2]吴艳.1359例手足口病流行病学分析.中国热带医学,2006(04)：607.
[3]Huang KY, Zhang XW, Chung PH, et al. Enterovirus 71 in Taiwan,2004-2006: epidemiological and virological features. Scand J Infect Dis,2008.40(6-7): 571-574.
[4]Ho M, Chen ER, Hsu KH, et al. An epidemic of enterovirus 71 infection in Taiwan. Taiwan Enterovirus Epidemic Working Group. N Engl J Med,1999. 341(13):929-935.
[5]Nolan MA,Craig MM,Lahra WD, et al. Survival after pulmonary edema due to enterovirus 71 encephalitis. Neurology,2003.60(10):1651-1656.
[6]Chang, LY,Huang LM, Hsia SH, et al. Neurodevelopment and cognition in children after enterovirus 71 infection. N Engl J Med,2007.356(12): 1226-1234.
[7]Lum, L.C, Wong KT, Lam SK, et al. Fatal enterovirus 71 encephalomyelitis. J Pediatr,1998.133(6):795-798.
[8]Brown, M.R. and A. Kornberg, Inorganic polyphosphate in the origin and survival of species. Proc Natl Acad Sci U S A,2004.101(46):16085-16087.
[9]Brown, BA, Oberste MS, Alexander JP, et al. Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to 1998. J Virol,1999. 73(12):9969-9975.
[10]Cardosa, MJ, Perera D, Brown BA, et al. Molecular epidemiology of human enterovirus 71 strains and recent outbreaks in the Asia-Pacific region: comparative analysis of the VP1 and VP4 genes. Emerg Infect Dis,2003.9(4): 461-468.
[11]McMinn, P, Llindsav K, Perera D, et al. Phylogenetic analysis of enterovirus 71 strains isolated during linked epidemics in Malaysia, Singapore, and Western Australia. J Virol,2001.75(16):7732-7738.
[12]Solomon T, Lewthwaite P, Perera D, et al. Virology, epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis,2010.10(11): 778-790.
[13]McMinn P, Stratoy I, Nagaraian L, et al.Neurological manifestations of enterovirus 71 infection in children during an outbreak of hand, foot, and mouth disease in Western Australia. Clin Infect Dis,2001.32(2):236-242.
[14]Shih, S.R, Li YS, Chiou CC, et al. Expression of capsid [correction of caspid] protein VP1 for use as antigen for the diagnosis of enterovirus 71 infection. J Med Virol,2000.61(2):228-234.
[15]Lum, LC, Wong KT, Lam SK, et al. Neurogenic pulmonary oedema and enterovirus 71 encephalomyelitis. Lancet,1998.352(9137):1391.
[16]林馨和韩关根.肠道病毒71型分子生物学研究.浙江中医药大学学报,2008(03)：289-292.
[17]刘丽艳和朱启镕.肠道病毒71型分子生物学研究进展.微生物与感染,2010(01)：57-60.
[18]Schmidt NJ, Lennette EH, and Ho HH. An apparently new enterovirus isolated from patients with disease of the central nervous system. J Infect Dis,1974. 129(3):304-309.
[19]周世力,李琳琳,何雅青等.我国分离的肠道病毒71型(SHZH03病毒株)全基因组核苷酸序列分析.病毒学报,2004(01)：7-11.
[20]林思恩,章青,谢华萍等.我国广东、福建地区2000～2001年手足口病肠道病毒71型分离株的种系进化分析.中华实验和临床病毒学杂志,2004(03)：28-30.
[21]崔爱利,许文波,李秀珠.肠道病毒71型的RT-PCR诊断及基因特征.病毒学报,2004(02)：160-165.
[22]雷永良,王晓光,陈秀英.肠道病毒71型浙江毒株全基因组核苷酸序列测定分析.中国卫生检验杂志,2009(01)：30-33.
[23]Huang CC, Liu CC, Chang YC, et al. Neurologic complications in children with enterovirus 71 infection. N Engl J Med,1999.341(13):936-942.
[24]Gau SS, Chang LY, Huang LM, et al. Attention-deficit/hyperactivity-related symptoms among children with enterovirus 71 infection of the central nervous system. Pediatrics,2008.122(2):452-458.
[25]Yang F, Ren L, Xiong ZH, et al. Enterovirus 71 outbreak in the People's Republic of China in 2008. J Clin Microbiol,2009.47(7):2351-2352.
[26]Mizuta K, Abiko C, Murata T, et al. Frequent importation of enterovirus 71 from surrounding countries into the local community of Yamagata, Japan, between 1998 and 2003. J Clin Microbiol,2005.43(12):6171-6175.
[27]Kung CM, King CC, Lee CN, et al. Differences in replication capacity between enterovirus 71 isolates obtained from patients with encephalitis and those obtained from patients with herpangina in Taiwan. J Med Virol,2007.79(1): 60-68.
[28]Komatsu H, Shimizu Y, Takeuchi Y, et al. Outbreak of severe neurologic involvement associated with Enterovirus 71 infection. Pediatr Neurol,1999. 20(1):17-23.
[29]Feigin, I. Lesions in the white matter in acute poliomyelitis. Neurology,1957. 7(6):399-403.
[30]董晓楠,应剑,陈应华.1970-2004年全球肠道病毒71型分离株的分子流行病学分析.科学通报,2007(09)：1021-1027.
[31]Shieh WJ, Jung SM, Hsueh C, et al. Pathologic studies of fatal cases in outbreak of hand, foot, and mouth disease, Taiwan. Emerg Infect Dis,2001. 7(1):146-148.
[32]Yang Y, Wang H, Gong E, et al. Neuropathology in 2 cases of fatal enterovirus type 71 infection from a recent epidemic in the People's Republic of China:a histopathologic, immunohistochemical, and reverse transcription polymerase chain reaction study. Hum Pathol,2009.40(9):1288-1295.
[33]Crotty S, Hix L, Sigal LJ, et al. Poliovirus pathogenesis in a new poliovirus receptor transgenic mouse model:age-dependent paralysis and a mucosal route of infection. J Gen Virol, 2002.83(Pt 7):1707-1720.
[34]Ohka S, Matsuda N, Tohyama K, et al. Receptor (CD155)-dependent endocytosis of poliovirus and retrograde axonal transport of the endosome. J Virol,2004.78(13):7186-7198.
[35]彭镜,尹飞,甘娜.体外血脑屏障模型的建立.中国当代儿科杂志,2005(06)：526-529.
[36]Pandey, A. and M. Mann, Proteomics to study genes and genomes. Nature, 2000.405(6788):837-846.
[37]武士京,陈世畯.血脑屏障——解剖与生理.现代神经疾病杂志,2003(02)：124-128.
[38]贾文祥.医学微生物学.2001.75.
[39]杨绍基.卫生部发布2008年版“手足口病预防控制指南”.新医学,2008(06)：363.
[40]中国CDC,手足口病标本采集及检测技术方案.2009.
[41]Zhang, K, Zhao WD, Li Q, et al. Tentative identification of glycerol dehydrogenase as Escherichia coli K1 virulence factor cglD and its involvement in the pathogenesis of experimental neonatal meningitis. Med Microbiol Immunol,2009.198(3):195-204.
[42]常宏伟,张文艳,范玉珍等.2010年六安市肠道病毒71型的分子流行病学特征.中国卫生检验杂志,2011(10)：2364-2367.
[43]Abbott NJ. Astrocyte-endothelial interactions and blood-brain barrier permeability. J Anat,2002.200(6):629-638.
[44]Afonso PV, Ozden S, Cumont MC, et al. Alteration of blood-brain barrier integrity by retroviral infection. PloS Pathog,2008.4(11):el000205.
[45]Stins MF, Badgern J and Kim KS. Bacterial invasion and transcytosis in transfected human brain microvascular endothelial cells. Microb Pathog,2001. 30(1):19-28.
[46]Lin YW, Lin HY, Tsou YL,et al. Human SCARB2-mediated entry and endocytosis of EV71. PloS One,2012.7(1):30507.
[47]Chen TC, Lai YK, Yu CK, et al. Enterovirus 71 triggering of neuronal apoptosis through activation of Abl-Cdk5 signalling. Cell Microbiol,2007. 9(11):2676-2688.
[48]董小曼,田博,董翊等.,滴定痘苗病毒的噬斑血吸附法的优化.中国生物制品学杂志,2007(11)：855-857.
[49]蒋维.肠道病毒71型病毒样颗粒的制备、纯化与鉴定.2010,北京工业大学.
[50]Luftig RB. Does the cytoskeleton play a significant role in animal virus replication? J Theor Biol,1982.99(1):173-191.
[51]Galan JE and Zhou D. Striking a balance:modulation of the actin cytoskeleton by Salmonella. Proc Natl Acad Sci U S A,2000.97(16):8754-8761.
[52]Eugene E. Microvilli-like structures are associated with the internalization of virulent capsulated Neisseria meningitidis into vascular endothelial cells. J Cell Sci,2002.115(Pt6):1231-1241.
[53]Khan NA, Wang Y, Kim KJ, et al. Cytotoxic necrotizing factor-1 contributes to Escherichia coli K1 invasion of the central nervous system. J Biol Chem,2002. 277(18):15607-15612.
[54]Gupta, S. Involvement of actin microfilaments in the replication of human parainfluenza virus type 3. J Virol,1998.72(4):2655-2662.
[55]Anuradha CD, Kanno S and Hirano S. Oxidative damage to mitochondria is a preliminary step to caspase-3 activation in fluoride-induced apoptosis in HL-60 cells. Free Radic Biol Med,2001.31(3):367-373.
[56]Smaili SS, Hsu YT, Sanders KM, et al. Bax translocation to mitochondria subsequent to a rapid loss of mitochondrial membrane potential. Cell Death Differ,2001.8(9):909-920.
[57]Fowlkes AL, Honarmand S, Glaser carol, et al. Enterovirus-associated encephalitis in the California encephalitis project,1998-2005. J Infect Dis, 2008.198(11):1685-1691.
[58]Hamaguchi T, Fujisawa H and Sakai kj, et al. Acute encephalitis caused by intrafamilial transmission of enterovirus 71 in adult. Emerg Infect Dis,2008. 14(5):828-830.
[59]McMinn PC. An overview of the evolution of enterovirus 71 and its clinical and public health significance. FEMS Microbiol Rev,2002.26(1):91-107.
[60]Singh S, Hsu YT,Sanders KM, et al. Direct detection of enterovirus 71 (EV71) in clinical specimens from a hand, foot, and mouth disease outbreak in Singapore by reverse transcription-PCR with universal enterovirus and EV71-specific primers. J Clin Microbiol,2002.40(8):2823-2827.
[61]Huang, S.W., et al., Reemergence of enterovirus 71 in 2008 in taiwan: dynamics of genetic and antigenic evolution from 1998 to 2008. J Clin Microbiol,2009.47(11):3653-3662.
[62]Yamayoshi S, Yamashita Y and Li j, et al. Scavenger receptor B2 is a cellular receptor for enterovirus 71. Nat Med,2009.15(7):798-801.
[63]郑焕英.人肠道病毒感染及其研究进展.在新发和再发传染病防治热点研讨会.2010.中国广东广州.
[64]Yang WX, Terasaki T, Shiroki K, et al. Efficient delivery of circulating poliovirus to the central nervous system independently of poliovirus receptor. Virology,1997.229(2):421-428.
[65]Racaniello VR. One hundred years of poliovirus pathogenesis. Virology,2006. 344(1):9-16.
[66]Liang CC, Sun MJ and Lei HY, et al. Human endothelial cell activation and apoptosis induced by enterovirus 71 infection. J Med Virol,2004.74(4): 597-603.
[67]矫毓娟和刘江红.细胞凋亡的检测方法(综述).中国神经免疫学和神经病学杂志,2004(01)：53-56.
[68]Lemasters JJ. Selective mitochondrial autophagy, or mitophagy, as a targeted defense against oxidative stress, mitochondrial dysfunction, and aging. Rejuvenation Res,2005.8(1):3-5.
[69]赵广圣和林茂芳.巨细胞病毒感染的细胞微丝骨架重组异常.中国实验血液学杂志,2006(04)：759-762.
[70]Huang HI, Weng KF and Shih SR. Viral and host factors that contribute to pathogenicity of enterovirus 71. Future Microbiol,2012.7:467-479.
[71]Kavallaris M and Marshall GM. Proteomics and disease:opportunities and challenges. Med J Aust,2005.182(11):575-579.
[72]宋革和姜勇.二维凝胶电泳的新技术及其应用.中国微循环,2005(01)：62-65.
[73]Brown JN, Palermo RE, Baskin CR, et al. Macaque proteome response to highly pathogenic avian influenza and 1918 reassortant influenza virus infections. J Virol,2010.84(22):12058-12068.
[74]Zheng X, Hong LL, Shi LX, et al. Proteomics analysis of host cells infected with infectious bursal disease virus. Mol Cell Proteomics,2008.7(3):612-625.
[75]Liu N, Wenjun S and Pui W, et al. Proteomics analysis of differential expression of cellular proteins in response to avian H9N2 virus infection in human cells. Proteomics,2008.8(9):1851-1858.
[76]Zhao C, Fang CY, Tian XC, et al. Proteomic analysis of hepatitis B surface antigen positive transgenic mouse liver and decrease of cyclophilin A. J Med Virol,2007.79(10):1478-1484.
[77]Sinha S, Arora S and Kosaiai K, et al. Proteome analysis of the plasma membrane of Mycobacterium tuberculosis. Comp Funct Genomics,2002.3(6): 470-483.
[78]贺福初和万晶宏,疾病蛋白质组学研究进展.中国医学科学院学报,2002(03)：219-222.
[79]Roepstorff P. Mass spectrometry in protein studies from genome to function. Curr Opin Biotechnol,1997.8(1):6-13.
[80]Henzel WJ. Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc Natl Acad Sci US A,1993.90(11):5011-5015.
[81]万晶宏和贺福初.蛋白质组技术的研究进展.科学通报,1999(09)：904-911.
[82]成海平和钱小红.蛋白质组研究的技术体系及其进展.生物化学与生物物理进展,2000(06)：584-588.
[83]Mann M, Hendrickson RC and Pandey A. Analysis of proteins and proteomes by mass spectrometry. Annu Rev Biochem,2001.70:437-473.
[84]Yates JR.3rd, Mass spectrometry and the age of the proteome. J Mass Spectrom,1998.33(1):1-19.
[85]李峰,肖志强,陈主初.质谱数据库查询软件的应用与评价.生命的化学,2003(06)：467-469.
[86]葛文松.乙型肝炎病毒相关性肝纤维化组织的比较蛋白质组学研究.2007,第二军医大学.
[87]Bienvenut WV. Matrix-assisted laser desorption/ionization-tandem mass spectrometry with high resolution and sensitivity for identification and characterization of proteins. Proteomics,2002.2(7):868-876.
[88]谢朝晖和陈兰英.细胞骨架研究进展.癌变·畸变·突变,2011(04)：315-318.
[89]Bernard O. Lim kinases, regulators of actin dynamics. Int J Biochem Cell Biol, 2007.39(6):1071-1076.
[90]Bernstein BW and Bamburg JR. ADF/cofilin:a functional node in cell biology. Trends Cell Biol,2010.20(4):187-195.
[91]陈炜,王嘉丽,彭涛等.微管骨架在登革病毒感染ECV304细胞中作用的初步研究.第三军医大学学报,2005(07)：628-631.
[92]Liu WJ. Association of bovine papillomavirus type 1 with microtubules. Virology,2001.282(2):237-244.
[93]Sodeik B, Ebersold MW and Helenius A. Microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus. J Cell Biol,1997. 136(5):1007-1021.
[94]Ward BM and Moss B. Vaccinia virus intracellular movement is associated with microtubules and independent of actin tails. J Virol.2001.75(23): 11651-11663.
[95]Schaffeld M, Herrmann H, Schultess J, et al. Vimentin and desmin of a cartilaginous fish, the shark Scyliorhinus stellaris:sequence, expression patterns and in vitro assembly. Eur J Cell Biol,2001.80(11):692-702.
[96]Sanjuan NA, Porras and Otero J. Microtubule-dependent intracellular transport of murine polyomavirus. Virology,2003.313(1):105-116.
[97]Toker A, Sellers LA, Amess B, et al. Multiple isoforms of a protein kinase C inhibitor (KCIP-1/14-3-3) from sheep brain. Amino acid sequence of phosphorylated forms. Eur J Biochem,1992.206(2):453-461.
[98]Ichimura T, Isobe T, Okuvama T, et al. Molecular cloning of cDNA coding for brain-specific 14-3-3 protein, a protein kinase-dependent activator of tyrosine and tryptophan hydroxylases. Proc Natl Acad Sci U S A,1988.85(19): 7084-7088.
[99]Aitken A.14-3-3 alpha and delta are the phosphorylated forms of raf-activating 14-3-3 beta and zeta. In vivo stoichiometric phosphorylation in brain at a Ser-Pro-Glu-Lys MOTIF. J Biol Chem,1995.270(11):5706-5709.
[100]Bridges D and Moorhead GB.14-3-3 proteins:a number of functions for a numbered protein. Sci STKE,2005.2005(296):10.
[101]Morimoto RI. Cells in stress:transcriptional activation of heat shock genes. Science,1993.259(5100):1409-1410.
[102]Coulombe PA and Wong P. Cytoplasmic intermediate filaments revealed as dynamic and multipurpose scaffolds. Nat Cell Biol,2004.6(8):699-706.
[103]Singh S, Sadacharan S, Su S, et al. Overexpression of vimentin:role in the invasive phenotype in an androgen-independent model of prostate cancer. Cancer Res,2003.63(9):2306-2311.
[104]Byun, Y. Caspase cleavage of vimentin disrupts intermediate filaments and promotes apoptosis. Cell Death Differ,2001.8(5):443-450.
[105]Kreplak, L. and D. Fudge, Biomechanical properties of intermediate filaments: from tissues to single filaments and back. Bioessays,2007.29(1):26-35.
[106]Lorch H,Weiger D, Wagner V, et al. Current practice of temporary vena cava filter insertion:a multicenter registry. J Vasc Interv Radiol,2000.11(1):p. 83-8.
[107]Fuchs E. Intermediate filaments and disease:mutations that cripple cell strength. J Cell Biol,1994.125(3):511-516.
[108]Styers ML, Salazar G, Love R, et al. The endo-lysosomal sorting machinery interacts with the intermediate filament cytoskeleton. Mol Biol Cell,2004. 15(12):5369-5382.
[109]Herrmann H and Aebi U. Intermediate filaments and their associates: multi-talented structural elements specifying cytoarchitecture and cytodynamics. Curr Opin Cell Biol,2000.12(1):79-90.
[2]吴艳.1359例手足口病流行病学分析.中国热带医学,2006(04)：607.
[3]Huang KY, Zhang XW, Chung PH, et al. Enterovirus 71 in Taiwan,2004-2006: epidemiological and virological features. Scand J Infect Dis,2008.40(6-7): 571-574.
[4]Ho M, Chen ER, Hsu KH, et al. An epidemic of enterovirus 71 infection in Taiwan. Taiwan Enterovirus Epidemic Working Group. N Engl J Med,1999. 341(13):929-935.
[5]Nolan MA,Craig MM,Lahra WD, et al. Survival after pulmonary edema due to enterovirus 71 encephalitis. Neurology,2003.60(10):1651-1656.
[6]Chang, LY,Huang LM, Hsia SH, et al. Neurodevelopment and cognition in children after enterovirus 71 infection. N Engl J Med,2007.356(12): 1226-1234.
[7]Lum, L.C, Wong KT, Lam SK, et al. Fatal enterovirus 71 encephalomyelitis. J Pediatr,1998.133(6):795-798.
[8]Brown, M.R. and A. Kornberg, Inorganic polyphosphate in the origin and survival of species. Proc Natl Acad Sci U S A,2004.101(46):16085-16087.
[9]Brown, BA, Oberste MS, Alexander JP, et al. Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to 1998. J Virol,1999. 73(12):9969-9975.
[10]Cardosa, MJ, Perera D, Brown BA, et al. Molecular epidemiology of human enterovirus 71 strains and recent outbreaks in the Asia-Pacific region: comparative analysis of the VP1 and VP4 genes. Emerg Infect Dis,2003.9(4): 461-468.
[11]McMinn, P, Llindsav K, Perera D, et al. Phylogenetic analysis of enterovirus 71 strains isolated during linked epidemics in Malaysia, Singapore, and Western Australia. J Virol,2001.75(16):7732-7738.
[12]Solomon T, Lewthwaite P, Perera D, et al. Virology, epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis,2010.10(11): 778-790.
[13]McMinn P, Stratoy I, Nagaraian L, et al.Neurological manifestations of enterovirus 71 infection in children during an outbreak of hand, foot, and mouth disease in Western Australia. Clin Infect Dis,2001.32(2):236-242.
[14]Shih, S.R, Li YS, Chiou CC, et al. Expression of capsid [correction of caspid] protein VP1 for use as antigen for the diagnosis of enterovirus 71 infection. J Med Virol,2000.61(2):228-234.
[15]Lum, LC, Wong KT, Lam SK, et al. Neurogenic pulmonary oedema and enterovirus 71 encephalomyelitis. Lancet,1998.352(9137):1391.
[16]林馨和韩关根.肠道病毒71型分子生物学研究.浙江中医药大学学报,2008(03)：289-292.
[17]刘丽艳和朱启镕.肠道病毒71型分子生物学研究进展.微生物与感染,2010(01)：57-60.
[18]Schmidt NJ, Lennette EH, and Ho HH. An apparently new enterovirus isolated from patients with disease of the central nervous system. J Infect Dis,1974. 129(3):304-309.
[19]周世力,李琳琳,何雅青等.我国分离的肠道病毒71型(SHZH03病毒株)全基因组核苷酸序列分析.病毒学报,2004(01)：7-11.
[20]林思恩,章青,谢华萍等.我国广东、福建地区2000～2001年手足口病肠道病毒71型分离株的种系进化分析.中华实验和临床病毒学杂志,2004(03)：28-30.
[21]崔爱利,许文波,李秀珠.肠道病毒71型的RT-PCR诊断及基因特征.病毒学报,2004(02)：160-165.
[22]雷永良,王晓光,陈秀英.肠道病毒71型浙江毒株全基因组核苷酸序列测定分析.中国卫生检验杂志,2009(01)：30-33.
[23]Huang CC, Liu CC, Chang YC, et al. Neurologic complications in children with enterovirus 71 infection. N Engl J Med,1999.341(13):936-942.
[24]Gau SS, Chang LY, Huang LM, et al. Attention-deficit/hyperactivity-related symptoms among children with enterovirus 71 infection of the central nervous system. Pediatrics,2008.122(2):452-458.
[25]Yang F, Ren L, Xiong ZH, et al. Enterovirus 71 outbreak in the People's Republic of China in 2008. J Clin Microbiol,2009.47(7):2351-2352.
[26]Mizuta K, Abiko C, Murata T, et al. Frequent importation of enterovirus 71 from surrounding countries into the local community of Yamagata, Japan, between 1998 and 2003. J Clin Microbiol,2005.43(12):6171-6175.
[27]Kung CM, King CC, Lee CN, et al. Differences in replication capacity between enterovirus 71 isolates obtained from patients with encephalitis and those obtained from patients with herpangina in Taiwan. J Med Virol,2007.79(1): 60-68.
[28]Komatsu H, Shimizu Y, Takeuchi Y, et al. Outbreak of severe neurologic involvement associated with Enterovirus 71 infection. Pediatr Neurol,1999. 20(1):17-23.
[29]Feigin, I. Lesions in the white matter in acute poliomyelitis. Neurology,1957. 7(6):399-403.
[30]董晓楠,应剑,陈应华.1970-2004年全球肠道病毒71型分离株的分子流行病学分析.科学通报,2007(09)：1021-1027.
[31]Shieh WJ, Jung SM, Hsueh C, et al. Pathologic studies of fatal cases in outbreak of hand, foot, and mouth disease, Taiwan. Emerg Infect Dis,2001. 7(1):146-148.
[32]Yang Y, Wang H, Gong E, et al. Neuropathology in 2 cases of fatal enterovirus type 71 infection from a recent epidemic in the People's Republic of China:a histopathologic, immunohistochemical, and reverse transcription polymerase chain reaction study. Hum Pathol,2009.40(9):1288-1295.
[33]Crotty S, Hix L, Sigal LJ, et al. Poliovirus pathogenesis in a new poliovirus receptor transgenic mouse model:age-dependent paralysis and a mucosal route of infection. J Gen Virol, 2002.83(Pt 7):1707-1720.
[34]Ohka S, Matsuda N, Tohyama K, et al. Receptor (CD155)-dependent endocytosis of poliovirus and retrograde axonal transport of the endosome. J Virol,2004.78(13):7186-7198.
[35]彭镜,尹飞,甘娜.体外血脑屏障模型的建立.中国当代儿科杂志,2005(06)：526-529.
[36]Pandey, A. and M. Mann, Proteomics to study genes and genomes. Nature, 2000.405(6788):837-846.
[37]武士京,陈世畯.血脑屏障——解剖与生理.现代神经疾病杂志,2003(02)：124-128.
[38]贾文祥.医学微生物学.2001.75.
[39]杨绍基.卫生部发布2008年版“手足口病预防控制指南”.新医学,2008(06)：363.
[40]中国CDC,手足口病标本采集及检测技术方案.2009.
[41]Zhang, K, Zhao WD, Li Q, et al. Tentative identification of glycerol dehydrogenase as Escherichia coli K1 virulence factor cglD and its involvement in the pathogenesis of experimental neonatal meningitis. Med Microbiol Immunol,2009.198(3):195-204.
[42]常宏伟,张文艳,范玉珍等.2010年六安市肠道病毒71型的分子流行病学特征.中国卫生检验杂志,2011(10)：2364-2367.
[43]Abbott NJ. Astrocyte-endothelial interactions and blood-brain barrier permeability. J Anat,2002.200(6):629-638.
[44]Afonso PV, Ozden S, Cumont MC, et al. Alteration of blood-brain barrier integrity by retroviral infection. PloS Pathog,2008.4(11):el000205.
[45]Stins MF, Badgern J and Kim KS. Bacterial invasion and transcytosis in transfected human brain microvascular endothelial cells. Microb Pathog,2001. 30(1):19-28.
[46]Lin YW, Lin HY, Tsou YL,et al. Human SCARB2-mediated entry and endocytosis of EV71. PloS One,2012.7(1):30507.
[47]Chen TC, Lai YK, Yu CK, et al. Enterovirus 71 triggering of neuronal apoptosis through activation of Abl-Cdk5 signalling. Cell Microbiol,2007. 9(11):2676-2688.
[48]董小曼,田博,董翊等.,滴定痘苗病毒的噬斑血吸附法的优化.中国生物制品学杂志,2007(11)：855-857.
[49]蒋维.肠道病毒71型病毒样颗粒的制备、纯化与鉴定.2010,北京工业大学.
[50]Luftig RB. Does the cytoskeleton play a significant role in animal virus replication? J Theor Biol,1982.99(1):173-191.
[51]Galan JE and Zhou D. Striking a balance:modulation of the actin cytoskeleton by Salmonella. Proc Natl Acad Sci U S A,2000.97(16):8754-8761.
[52]Eugene E. Microvilli-like structures are associated with the internalization of virulent capsulated Neisseria meningitidis into vascular endothelial cells. J Cell Sci,2002.115(Pt6):1231-1241.
[53]Khan NA, Wang Y, Kim KJ, et al. Cytotoxic necrotizing factor-1 contributes to Escherichia coli K1 invasion of the central nervous system. J Biol Chem,2002. 277(18):15607-15612.
[54]Gupta, S. Involvement of actin microfilaments in the replication of human parainfluenza virus type 3. J Virol,1998.72(4):2655-2662.
[55]Anuradha CD, Kanno S and Hirano S. Oxidative damage to mitochondria is a preliminary step to caspase-3 activation in fluoride-induced apoptosis in HL-60 cells. Free Radic Biol Med,2001.31(3):367-373.
[56]Smaili SS, Hsu YT, Sanders KM, et al. Bax translocation to mitochondria subsequent to a rapid loss of mitochondrial membrane potential. Cell Death Differ,2001.8(9):909-920.
[57]Fowlkes AL, Honarmand S, Glaser carol, et al. Enterovirus-associated encephalitis in the California encephalitis project,1998-2005. J Infect Dis, 2008.198(11):1685-1691.
[58]Hamaguchi T, Fujisawa H and Sakai kj, et al. Acute encephalitis caused by intrafamilial transmission of enterovirus 71 in adult. Emerg Infect Dis,2008. 14(5):828-830.
[59]McMinn PC. An overview of the evolution of enterovirus 71 and its clinical and public health significance. FEMS Microbiol Rev,2002.26(1):91-107.
[60]Singh S, Hsu YT,Sanders KM, et al. Direct detection of enterovirus 71 (EV71) in clinical specimens from a hand, foot, and mouth disease outbreak in Singapore by reverse transcription-PCR with universal enterovirus and EV71-specific primers. J Clin Microbiol,2002.40(8):2823-2827.
[61]Huang, S.W., et al., Reemergence of enterovirus 71 in 2008 in taiwan: dynamics of genetic and antigenic evolution from 1998 to 2008. J Clin Microbiol,2009.47(11):3653-3662.
[62]Yamayoshi S, Yamashita Y and Li j, et al. Scavenger receptor B2 is a cellular receptor for enterovirus 71. Nat Med,2009.15(7):798-801.
[63]郑焕英.人肠道病毒感染及其研究进展.在新发和再发传染病防治热点研讨会.2010.中国广东广州.
[64]Yang WX, Terasaki T, Shiroki K, et al. Efficient delivery of circulating poliovirus to the central nervous system independently of poliovirus receptor. Virology,1997.229(2):421-428.
[65]Racaniello VR. One hundred years of poliovirus pathogenesis. Virology,2006. 344(1):9-16.
[66]Liang CC, Sun MJ and Lei HY, et al. Human endothelial cell activation and apoptosis induced by enterovirus 71 infection. J Med Virol,2004.74(4): 597-603.
[67]矫毓娟和刘江红.细胞凋亡的检测方法(综述).中国神经免疫学和神经病学杂志,2004(01)：53-56.
[68]Lemasters JJ. Selective mitochondrial autophagy, or mitophagy, as a targeted defense against oxidative stress, mitochondrial dysfunction, and aging. Rejuvenation Res,2005.8(1):3-5.
[69]赵广圣和林茂芳.巨细胞病毒感染的细胞微丝骨架重组异常.中国实验血液学杂志,2006(04)：759-762.
[70]Huang HI, Weng KF and Shih SR. Viral and host factors that contribute to pathogenicity of enterovirus 71. Future Microbiol,2012.7:467-479.
[71]Kavallaris M and Marshall GM. Proteomics and disease:opportunities and challenges. Med J Aust,2005.182(11):575-579.
[72]宋革和姜勇.二维凝胶电泳的新技术及其应用.中国微循环,2005(01)：62-65.
[73]Brown JN, Palermo RE, Baskin CR, et al. Macaque proteome response to highly pathogenic avian influenza and 1918 reassortant influenza virus infections. J Virol,2010.84(22):12058-12068.
[74]Zheng X, Hong LL, Shi LX, et al. Proteomics analysis of host cells infected with infectious bursal disease virus. Mol Cell Proteomics,2008.7(3):612-625.
[75]Liu N, Wenjun S and Pui W, et al. Proteomics analysis of differential expression of cellular proteins in response to avian H9N2 virus infection in human cells. Proteomics,2008.8(9):1851-1858.
[76]Zhao C, Fang CY, Tian XC, et al. Proteomic analysis of hepatitis B surface antigen positive transgenic mouse liver and decrease of cyclophilin A. J Med Virol,2007.79(10):1478-1484.
[77]Sinha S, Arora S and Kosaiai K, et al. Proteome analysis of the plasma membrane of Mycobacterium tuberculosis. Comp Funct Genomics,2002.3(6): 470-483.
[78]贺福初和万晶宏,疾病蛋白质组学研究进展.中国医学科学院学报,2002(03)：219-222.
[79]Roepstorff P. Mass spectrometry in protein studies from genome to function. Curr Opin Biotechnol,1997.8(1):6-13.
[80]Henzel WJ. Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc Natl Acad Sci US A,1993.90(11):5011-5015.
[81]万晶宏和贺福初.蛋白质组技术的研究进展.科学通报,1999(09)：904-911.
[82]成海平和钱小红.蛋白质组研究的技术体系及其进展.生物化学与生物物理进展,2000(06)：584-588.
[83]Mann M, Hendrickson RC and Pandey A. Analysis of proteins and proteomes by mass spectrometry. Annu Rev Biochem,2001.70:437-473.
[84]Yates JR.3rd, Mass spectrometry and the age of the proteome. J Mass Spectrom,1998.33(1):1-19.
[85]李峰,肖志强,陈主初.质谱数据库查询软件的应用与评价.生命的化学,2003(06)：467-469.
[86]葛文松.乙型肝炎病毒相关性肝纤维化组织的比较蛋白质组学研究.2007,第二军医大学.
[87]Bienvenut WV. Matrix-assisted laser desorption/ionization-tandem mass spectrometry with high resolution and sensitivity for identification and characterization of proteins. Proteomics,2002.2(7):868-876.
[88]谢朝晖和陈兰英.细胞骨架研究进展.癌变·畸变·突变,2011(04)：315-318.
[89]Bernard O. Lim kinases, regulators of actin dynamics. Int J Biochem Cell Biol, 2007.39(6):1071-1076.
[90]Bernstein BW and Bamburg JR. ADF/cofilin:a functional node in cell biology. Trends Cell Biol,2010.20(4):187-195.
[91]陈炜,王嘉丽,彭涛等.微管骨架在登革病毒感染ECV304细胞中作用的初步研究.第三军医大学学报,2005(07)：628-631.
[92]Liu WJ. Association of bovine papillomavirus type 1 with microtubules. Virology,2001.282(2):237-244.
[93]Sodeik B, Ebersold MW and Helenius A. Microtubule-mediated transport of incoming herpes simplex virus 1 capsids to the nucleus. J Cell Biol,1997. 136(5):1007-1021.
[94]Ward BM and Moss B. Vaccinia virus intracellular movement is associated with microtubules and independent of actin tails. J Virol.2001.75(23): 11651-11663.
[95]Schaffeld M, Herrmann H, Schultess J, et al. Vimentin and desmin of a cartilaginous fish, the shark Scyliorhinus stellaris:sequence, expression patterns and in vitro assembly. Eur J Cell Biol,2001.80(11):692-702.
[96]Sanjuan NA, Porras and Otero J. Microtubule-dependent intracellular transport of murine polyomavirus. Virology,2003.313(1):105-116.
[97]Toker A, Sellers LA, Amess B, et al. Multiple isoforms of a protein kinase C inhibitor (KCIP-1/14-3-3) from sheep brain. Amino acid sequence of phosphorylated forms. Eur J Biochem,1992.206(2):453-461.
[98]Ichimura T, Isobe T, Okuvama T, et al. Molecular cloning of cDNA coding for brain-specific 14-3-3 protein, a protein kinase-dependent activator of tyrosine and tryptophan hydroxylases. Proc Natl Acad Sci U S A,1988.85(19): 7084-7088.
[99]Aitken A.14-3-3 alpha and delta are the phosphorylated forms of raf-activating 14-3-3 beta and zeta. In vivo stoichiometric phosphorylation in brain at a Ser-Pro-Glu-Lys MOTIF. J Biol Chem,1995.270(11):5706-5709.
[100]Bridges D and Moorhead GB.14-3-3 proteins:a number of functions for a numbered protein. Sci STKE,2005.2005(296):10.
[101]Morimoto RI. Cells in stress:transcriptional activation of heat shock genes. Science,1993.259(5100):1409-1410.
[102]Coulombe PA and Wong P. Cytoplasmic intermediate filaments revealed as dynamic and multipurpose scaffolds. Nat Cell Biol,2004.6(8):699-706.
[103]Singh S, Sadacharan S, Su S, et al. Overexpression of vimentin:role in the invasive phenotype in an androgen-independent model of prostate cancer. Cancer Res,2003.63(9):2306-2311.
[104]Byun, Y. Caspase cleavage of vimentin disrupts intermediate filaments and promotes apoptosis. Cell Death Differ,2001.8(5):443-450.
[105]Kreplak, L. and D. Fudge, Biomechanical properties of intermediate filaments: from tissues to single filaments and back. Bioessays,2007.29(1):26-35.
[106]Lorch H,Weiger D, Wagner V, et al. Current practice of temporary vena cava filter insertion:a multicenter registry. J Vasc Interv Radiol,2000.11(1):p. 83-8.
[107]Fuchs E. Intermediate filaments and disease:mutations that cripple cell strength. J Cell Biol,1994.125(3):511-516.
[108]Styers ML, Salazar G, Love R, et al. The endo-lysosomal sorting machinery interacts with the intermediate filament cytoskeleton. Mol Biol Cell,2004. 15(12):5369-5382.
[109]Herrmann H and Aebi U. Intermediate filaments and their associates: multi-talented structural elements specifying cytoarchitecture and cytodynamics. Curr Opin Cell Biol,2000.12(1):79-90.