稳定表达鼠源整联蛋白αvβ6的CHO-677细胞系的构建

口蹄疫是由口蹄疫病毒(FMDV)引起的一种高度接触性传染病,主要侵害偶蹄动物。乳鼠常作为一种重要的实验动物模型用于FMDV的研究;整联蛋白αvβ6是FMDV的重要受体之一。为深入研究整联蛋白αvβ6在FMDV感染乳鼠中所发挥的作用,克隆了乳鼠整联蛋白αvβ6的两个亚基,并将其导入中国仓鼠卵巢细胞(Chinese hamster ovary,CHO-677)基因组中,构建了稳定表达乳鼠整联蛋白αv和β6亚基的细胞系CHO-677-mαvβ6,并分别选用两种不同血清型的野生型FMDV毒株Asia1/HN/CHA/06和O/BY/CHA/2010感染细胞系来分析细胞系对FMDV的易感性。首先通过PCR和间接免疫荧光试验证明了细胞系中整联蛋白αvβ6在基因水平成功导入,在蛋白水平成功表达。然后,通过实时荧光定量RT-PCR检测病毒RNA拷贝数,并结合TCID50试验测定了代表毒株在两个细胞上的生长曲线。结果表明,与亲本细胞CHO-677相比,细胞系CHO-677-mαvβ6对FMDV更易感,从αvβ6的功能性上进一步验证了细胞系被成功构建。     

口蹄疫病毒受体的结构与功能研究进展

口蹄疫病毒（FMDV）感染过程中的特异性受体是FMDV识别,结合宿主细胞的分子基础,研究FMDV受体的结构与功能对防治口蹄疫具有重要的理论意义和应用价值。本论述了近年来关于整联蛋白αvβ3,αvβ6和硫酸乙酰肝素（HS）三种FMDV受体的结构与功能的研究进展。     

整联蛋白与口蹄疫病毒感染

整联蛋白是一类分布广泛的细胞表面受体家族,它不仅在细胞的生长、移行、增殖和分化等许多方面发挥重要生物学功能,而且在多种病理过程中发挥重要作用。许多病毒都能利用整联蛋白分子作为病毒受体或共受体进入宿主细胞。本文主要就整联蛋白的特征及其在口蹄疫病毒感染宿主细胞过程中的作用机制进行综述。     

口蹄疫病毒与宿主细胞的相互作用研究进展

口蹄疫病毒（FMDV）导致了偶蹄动物口蹄疫的发生,它是一类有着自身特点的RNA病毒。首先,FMDV衣壳蛋白VP1识别结合宿主细胞膜上的整联蛋白等受体,以内吞的方式进入细胞,利用宿主细胞成分完成病毒蛋白的合成。这些新合成的L^pro、2C和3C^pro等病毒致病因子进一步抑制宿主基因的转录和翻译,诱导细胞凋亡和白噬,并抑制干扰素介导的一系列先天性和获得性免疫反应。宿主则在病毒侵染细胞的初期,利用病毒识别受体等来识别病毒并诱导合成干扰素等细胞因子,介导多种免疫反应以清除病毒。病毒和宿主两者在持续的利用和较量中完成疾病的发生和痊愈等。其次,不断发现的病毒受体、结合基序、致病因子及宿主细胞的多种免疫调节因子将成为相关领域新的研究内容。综上,开发高效安全疫苗、增强自身免疫力及利用RNAi直接抑制病毒RNA等便成为现代FMDV防治的主要内容。     

整联蛋白调控血管新生内膜增生的研究进展

血管新生内膜增生是支架植入术、动静脉瘘术等血管手术以及动脉粥样硬化、高血压等心血管疾病的生理特征。整联蛋白介导的细胞黏附在新生内膜增生过程中起着重要作用。该文概述了整联蛋白在此过程中对白细胞黏附、平滑肌细胞迁移增殖、再内皮化的调控及目前用于研究新生内膜的相关动物模型。了解整联蛋白调节血管新生内膜增生的分子机制,为临床上防治新生内膜增生、解决术后血管再狭窄等相关研究提供参考。     

整联蛋白与其配体结合的分子机制研究进展

整联蛋白是细胞表面的主要膜受体,具有介导细胞与细胞基质间的黏附及病毒吸附细胞等功能。αⅠ结构域型整联蛋白和非αⅠ结构域型整联蛋白与配体结合的方式各异。对整联蛋白-配体复合物分子结构的研究表明,整联蛋白利用其配体结合位点的αMIDAS（依赖金属离子的吸附位点）、βMIDAS与各种类型配体结合。     

整联蛋白信号与银屑病发病发子机制

整联蛋白可激活酪氨酸激酶和维持生长因子的生物活性,实现细胞外基质－整联蛋白－细胞内的信号转导功能,调节细胞生长、分化、粘附等生理功能;在病理状态下,如银屑病整联蛋白表达异常,可介导角质形成细胞的过度增生、异常分化甚至炎症的发生。本文综述调节细胞基本行为的整联蛋白信号转导及其与银屑病分子发病机制的关系。     

整联蛋白与生长因子受体信号转导通路间的"串话"

整联蛋白是由α、β两种跨膜亚基组成的异源二聚体,是介导胞外基质（ＥＣＭ）粘附的主要细胞表面受体家族。整联蛋白介导的粘附作用参与调节多种细胞功能。近十年间对整联蛋白的研究已从发现其家族的新成员转至描述各种整联蛋白的具体功能,主要热点在于：１．整联蛋白是细胞迁移的关键效应分子;２．整联蛋白与生长因子受体合作促进细胞的增殖,其原因至少部分是由于锚定依赖在细胞周期中由Ｇ１到Ｓ期所起的作用;３．粘附是细胞退出细胞周期开始分化的必要条件;４．当粘附的组织细胞脱离ＥＣＭ时,则丧失了存活信号而发生凋亡［１］。近…     

炭疽毒素受体结构和功能研究进展

肿瘤血管内皮标志物8(TEMS)和毛细血管形态发生蛋白2(CMG2)是已知的两个炭疽毒素受体,它们的主要功能是当炭疽杆菌侵染细胞时介导炭疽毒素进入宿主细胞.这两个受体都是涉及到细胞外基质动态平衡的I型跨膜蛋白,并且都因为它们在血管生成或血管内皮细胞中表达增强而被发现.有研究发现TEM8能够调节内皮细胞迁移和血管形成,而CMG2则在内皮细胞增殖过程中起重要作用.进一步的研究显示它们与整联蛋白同源性较高,但它们确切的生理功能和作用机制尚不明确.本文中,主要讨论这两种蛋白的结构和它们作为炭疽毒素受体介导炭疽毒素进入细胞的分子机制,然后我们简单探讨一下TEM8在靶向肿瘤血管内皮细胞的抗血管生成和抗肿瘤疗法方面的研究进展,最后我们展望了下一步炭疽毒素受体研究的热点-它们的配体及生理功能研究.     

单颗粒示踪技术及其在病毒侵染机制研究中的应用

病毒侵染宿主细胞是一个复杂的动态过程,且涉及病毒与细胞多种成分的相互作用,病毒侵染机制研究对研制抗病毒药物以及病毒性疾病的预防和治疗至关重要。单颗粒示踪技术是可视化研究生物动态过程的重要工具,能够研究病毒在活细胞中的侵染行为,现已成为病毒侵染机制研究的有效手段。此文综述了单颗粒示踪技术中病毒标记物、标记方法、活细胞成像及分析进展,以流行性感冒病毒和人免疫缺陷病毒为例阐述了该技术在病毒侵染机制研究中的应用,对该技术的应用前景进行了展望。此文旨在为病毒侵染机制研究介绍新的技术手段、推动病毒学研究。     

口蹄疫病毒诱导宿主细胞凋亡的研究

本文报道了口蹄疫病毒（Foot-and-Mouth Disease Virus,FMDV）在体外诱导PK－15细胞凋亡的研究结果,采用Hoechst33258荧光探针、DNA凝胶电泳、脱氧核糖核酸转移酶介导的制品末端标记（TUNEL）技术均检测到了典型的细胞凋亡,结果显示：使用感染性滴度为4.8lgTCID50/mL的口蹄疫病毒感染PK－15细胞,在培养32h后,荧光探针检测呈现典型的凋亡细胞核固缩和梅花状碎裂核,并伴随有凋亡小体出现,调亡率约为20％;DNA凝胶电泳显示ladder梯带;末端标记检测到强绿色荧光标记物结合于凋亡细胞核上。研究结果提示：口蹄疫病毒可以在体外诱导宿主细胞凋亡,细胞凋亡是其致细胞病变死亡的重要途径之一。     

口蹄疫病毒诱导宿主细胞凋亡的研究

本文报道了口蹄疫病毒（Foot-and-Mouth Disease Virus,FMDV）在体外诱导PK-15细胞凋亡的研究结果。采用Hoechst33258荧光探针、DNA凝胶电泳、脱氧核糖核酸转移酶介导的缺口末端标记（TUNEL）技术均检测到了典型的细胞凋亡。结果显示使用感染性滴度为4.8lgTCID50/mL的口蹄疫病毒感染PK-15细胞,在培养32?h后荧光探针检测呈现典型的凋亡细胞核固缩和梅花状碎裂核,并伴随有凋亡小体出现,凋亡率约为20%;DNA凝胶电泳显示ladder梯带;末端标记检测到强绿色荧光标记物结合于凋亡细胞核上。研究结果提示口蹄疫病毒可以在体外诱导宿主细胞凋亡,细胞凋亡是其致细胞病变死亡的重要途径之一。     

The DEAD-Box RNA Helicase DDX1 Interacts with the Viral Protein 3D and Inhibits Foot-and-Mouth Disease Virus Replication

Foot-and-mouth disease virus(FMDV) can infect domestic and wild cloven-hoofed animals. The non-structural protein 3 D plays an important role in FMDV replication and pathogenesis. However, the interaction partners of 3 D, and the effects of those interactions on FMDV replication, remain incompletely elucidated. In the present study, using the yeast two-hybrid system, we identified a porcine cell protein, DEAD-box RNA helicase 1(DDX1), which interacted with FMDV 3 D. The DDX1-3 D interaction was further confirmed by co-immunoprecipitation experiments and an indirect immunofluorescence assay(IFA) in porcine kidney 15(PK-15) cells. DDX1 was reported to either inhibit or facilitate viral replication and regulate host innate immune responses. However, the roles of DDX1 during FMDV infection remain unclear. Our results revealed that DDX1 inhibited FMDV replication in an ATPase/helicase activity-dependent manner. In addition, DDX1 stimulated IFN-b activation in FMDV-infected cells. Together, our results expand the body of knowledge regarding the role of DDX1 in FMDV infection.     

Isolation and identification of bovine nasopharyngeal mucosal epithelial cells and establishment of cell models of acute infection by foot-and-mouth disease virus

Foot-and-mouth disease (FMD) commonly occurs via the respiratory tract, and bovine nasopharyngeal mucosal epithelial cells are the primary infection cells in cattle. The aim of the present study was to isolate and culture epithelial cells from the bovine nasopharyngeal mucosa in vitro using a mechanical separation method. The cells were expanded, established in continuous cell culture, and used for immunofluorescence cytochemistry and establishment of infection models. We detected pan-cytokeratin markers of bovine nasopharyngeal mucosal epithelial cells by immunofluorescence. Bovine nasopharyngeal mucosal epithelial cells were then infected with foot-and-mouth disease virus (FMDV) serum type O. RT-PCR demonstrated the successful establishment of acute FMDV infection in the cell models. This infection model provides the basis for clarification of the interaction between FMDV and host bovine nasopharyngeal mucosal epithelial cells in vitro.     

Foot-and-Mouth Disease Virus Nonstructural Protein 2C Interacts with Beclin1, Modulating Virus Replication

Foot-and-mouth disease virus (FMDV), the causative agent of foot-and-mouth disease, is an Apthovirus within the Picornaviridae family. Replication of the virus occurs in association with replication complexes that are formed by host cell membrane rearrangements. The largest viral protein in the replication complex, 2C, is thought to have multiple roles during virus replication. However, studies examining the function of FMDV 2C have been rather limited. To better understand the role of 2C in the process of virus replication, we used a yeast two-hybrid approach to identify host proteins that interact with 2C. We report here that cellular Beclin1 is a specific host binding partner for 2C. Beclin1 is a regulator of the autophagy pathway, a metabolic pathway required for efficient FMDV replication. The 2C-Beclin1 interaction was further confirmed by coimmunoprecipitation and confocal microscopy to actually occur in FMDV-infected cells. Overexpression of either Beclin1 or Bcl-2, another important autophagy factor, strongly affects virus yield in cell culture. The fusion of lysosomes to autophagosomes containing viral proteins is not seen during FMDV infection, a process that is stimulated by Beclin1; however, in FMDV-infected cells overexpressing Beclin1 this fusion occurs, suggesting that 2C would bind to Beclin1 to prevent the fusion of lysosomes to autophagosomes, allowing for virus survival. Using reverse genetics, we demonstrate here that modifications to the amino acids in 2C that are critical for interaction with Beclin1 are also critical for virus growth. These results suggest that interaction between FMDV 2C and host protein Beclin1 could be essential for virus replication.     

Extensive cell heterogeneity during persistent infection with foot-and-mouth disease virus.

Coevolution of viruses and the host cells occurred in BHK-21 cell cultures persistently infected with foot-and-mouth disease virus (FMDV) (J. C. de la Torre, E. Martínez-Salas, J. Diez, A. Villaverde, F. Gebauer, E. Rocha, M. Dávila, and E. Domingo, J. Virol. 62:2050-2058, 1988). In the present report we provide evidence of an extreme phenotypic heterogeneity of the cells, which was generated in the course of persistence. A total of 248 stable cell clones isolated from FMDV carrier cultures at early or late passages were analyzed. At least six distinct cell phenotypes were distinguished with regard to cell morphology, resistance to FMDV strain C-S8c1, and cell growth characteristics. No infectious FMDV or viral RNA was detected in variant cell clones, suggesting that the altered phenotypes were caused by inheritable cell modifications, selected in the course of persistence. Thus, the FMDV-BHK-21 carrier cell system must be described as a dynamic interaction between an evolving heterogeneous population of virus and multiple cell variants. We suggest that cell heterogeneity confers a selective advantage for long-term virus and cell survival by providing the cell population with a range of responses toward FMDV.