树突状细胞与HIV-1 辅助蛋白相互作用的体外研究——新的实验体系的建立与应用

树突状细胞（DC）是免疫系统中具有多种免疫功能的关键细胞，但在HIV/AIDS 患者 体内，虽有大量病毒及抗原存在，却不能有效地激发特异性免疫应答。最新的研究显示， HIV/AIDS 血源和性传染的途径虽有不同，但DC 均是早于T 细胞而最先遭受感染的“第一 靶细胞”。已知辅助蛋白（Nef、Rev、Tat、Vif、Vpr 和Vpu）是HIV-1 在宿主细胞内复制 和致病的根本因素。而哪种辅助蛋白、如何影响DC 功能，至今研究结果甚少，结论杂乱 不一。 本论文作为HIV-1 影响DC 功能研究的实验体系部分，旨在为系统比较研究6 个辅助 蛋白对DC 功能的调节和机理奠定基础。采用分子生物学和免疫学技术方法，把辅助蛋白 基因从DNA 和mRNA 水平导入目的细胞DC，在DC 前体及其分化、成熟的各个时相， 连续、动态地分析辅助蛋白对DC 特征性表面标志及免疫相关基因的表达水平、摄取和处 理抗原、激发和调控免疫应答等功能的影响，以期揭示HIV/AIDS 患者DC 功能明显异常 和失调的原因。 首先，分别将6 个辅助蛋白基因克隆到pEGFP-N2 和pCS2+表达载体，得到具有绿色 荧光蛋白融合基因的表达载体，将分别用于DNA 和mRNA 水平转染DC。再以K562 细胞 为模型建立mRNA 转染细胞的基本方法；以人外周血CD14 单核细胞为前体，建立了体外 “2＋2”快速诱导DC 的方法。最后，利用Amaxa 转染系统，从DNA 水平研究辅助蛋白对 DC 前体（单核细胞）的功能影响。发现单核细胞转染辅助蛋白与GFP 融合基因5h 后，胞 内蛋白大量表达，且能维持表达48h；其中Nef、Tat、Vpu、Rev、Vif、Vpr 表达效率分别 为35.42%、34.42%、43.42%、 17.07%、13.65%、10.29%；单核细胞转染基因后，表型 CD14、HLA-DR、CD80、CD83、CD86、DC-SIGN 没有明显的表达变化；转染Nef、Vpu、 Rev 辅助蛋白后，单核细胞有10％凋亡；Vpr 能抑制单核细胞IL-10 的分泌，Nef 能促进分 泌IL-6。 总之，通过构建辅助蛋白的表达载体，优化DC 的体外培养过程以及mRNA 转染方法， 并成功将辅助蛋白导入DC 前体，鉴定其表型和功能变化。为研究辅助蛋白影响DC 的功 能和机制建立了稳定而可行的实验系统。

A single amino acid defines cross-species reactivity of tree shrew (Tupaia belangeri) CD1d to human invariant natural killer T (iNKT) cells

P>The non-classical major histocompatibility complex (MHC) class I molecule CD1d presents lipid antigens to invariant natural killer T (iNKT) cells, which are an important part of the innate immune system. CD1d/iNKT systems are highly conserved in evoluti

SEROLOGICAL SURVEY OF A CAPTIVE MACAQUE COLONY IN CHINA FOR ANTIBODIES TO SIMIAN TYPE-D RETROVIRUSES

Sera from 510 macaques consisting of Macaca mulatta, Macaca assamensis, Macaca fascicularis, Macaca nemestrina, and Macaca arctoides were investigated for antibodies to simian AIDS type D retrovirus (SRV) by ELISA and Western blot with viral antigens purified from supernatants of SRV-1 infected cell cultures. Of these monkeys, 104 were seropositive by ELISA; only 23 were confirmed by Western blot. The true positive reaction to SRV was found in 15 of 463 (3.2%) M. mulatta and eight of eleven (72.7%) M. assamensis.

SECRETORY MONOCLONAL IGA ANTIBODY TO HUMAN SPERM PRODUCED BY GASTROINTESTINAL IMMUNIZATION INHIBITS HUMAN SPERM ACTIVITY AND MOUSE INVITRO FERTILIZATION

BALB/c mice were immunized intragastrically with human sperm. Cells from the Peyer's patches and spleens of the immunized mice were for the preparation of hybridomas secreting antisperm monoclonal IgA (mcIgA). The specific ratio of IgA-secreting cells in Peyer's patches was much higher than that in spleen. The binding site on human sperm of 9 of 19 mcIgA was in the post-acrosomal region using an immunofluorescent assay. Two of eight selected mcIgA caused strong human sperm agglutination and three of them produced significant inhibition of mouse in vitro fertilization. No mcIgA tested caused obvious human sperm immobilization or inhibited mouse in vivo fertilization. In vitro assembly of selected mcIgA in ascites with mouse secretory component (SC) caused no significant changes in effects on sperm function and in vitro fertilization. By use of Western blotting, dimer or higher polymers were demonstrated in all selected mcIgAs and corresponding protein antigens in 6 of 8 selected mcIgAs. These results suggest that human sperm function may be inhibited and fertilization rate reduced by specific secretory IgA to human sperm and that secretory immunity to protein antigens of human sperm could be induced by intragastrointestinal immunization.

人类白细胞抗原-DRB1与胃腺癌及其临床特征和幽门螺杆菌感染的相关性

目的　探讨人类白细胞抗原 (HLA) DRB1等位基因与胃腺癌及其临床特征和幽门螺杆菌(Hp)感染的关联性。方法　运用序列特异性引物聚合酶链反应和等位基因序列分析技术 ,检测无亲缘关系湖北省汉族健康人 136例、胃癌组 6 3例的HLA DRB1基因。内镜活检、Giemsa染色和 (或 )外周血ELISA检查胃黏膜Hp感染情况。SAS软件数据处理。 结果　HLA DRB10 90 1、12等位基因均与湖北省汉族人胃腺癌呈正相关 ;HLA DRB115等位基因则呈负相关。携带及非携带上述各等位基因患者 ,分

人类白细胞抗原DRB1和DQB1等位基因多态性与大肠癌的关联性

目的　探讨人类白细胞抗原HLA DRB1, DQB1等位基因与大肠癌遗传关联性。方法　运用序列特异性引物聚合酶链反应 ,结合等位基因序列分析 ,检测无亲缘关系的湖北籍汉族健康人136名、大肠癌组 5 4例患者的HLA DRB1、 DQB1基因。结果　大肠癌患者与正常人比较 ,HLA DRB1 0 90 1等位基因分布频率明显增高 (0 2 315、0 1397,P =0 0 33) ;而 DRB1 0 80X等位基因频率明显降低 (0 0 0 93、0 0 80 9,P =0 0 0 71)。两组间HLA

Establishment of a Chinese sturgeon Acipenser sinensis tail-fin cell line and its susceptibility to frog iridovirus

Chinese sturgeon Acipenser sinensis, a cartilaginous ganoid, is a 'living fossil' on a deeply isolated evolutionary branch. A cell line was established from Chinese sturgeon tail-fin tissue (CSTF) . These epithelial CSTF cells grew well in Dulbecco's modified Eagle's medium at 25 degrees C. Karyotypic analysis revealed a normal diploid karyotype with 2n = 264 and large numbers of punctate chromosomes. A strain of frog iridoviruses [Rana grylio virus (RGV)] was used to test the susceptibility of this cell line to infection. Infection was confirmed by cytopathic effect, immunofluorescence and electron-microscope observations, which detected the viral antigens or particles in the cytoplasm of RGV-infected cells. Molecular analysis further suggested that c. 550 bp DNA fragment could be cloned from the RGV-infected CSTF cells' DNA with major capsid protein gene polymerase chain reaction primers. Furthermore, after transfection with pEGFP vector DNA, the CSTF cell line produced significant fluorescent signals indicating its utility in exogenous studies.

Detection of cutaneous antibodies in excised skin explants from grass carp, Ctenopharyngodon idella (Valenciennes), immune to Scophthalmus maximus rhabdovirus

This study determined whether cutaneous antibodies were present in excised skin explants of grass carp, Ctenopharyngodon idella, immune to Scophthalmus maximus rhabdovirus (SMRV). Culture fluid from immune skin explants were assayed by indirect enzyme-linked immunosorbent assay (iELISA), Western blot, indirect immunofluorescent assay (IFA) and flow cytometry (FCM). iELISA showed that cutaneous antibody titres were much lower (1:12) than antiserum titres (1:1458) from intraperitoneally immunized grass carp. The phosphoprotein and matrix protein antigens of purified SMRV proteins were recognized by cutaneous antibodies from skin culture fluid using Western blot. The skin culture fluid produced staining signals in viral assembly sites and cytoplasm of SMRV-infected epithelioma papulosum cyprini (EPC) cells by IFA. FCM showed that 4.39% SMRV-infected EPC cells were detected, while non-specific reaction was seen in 2% of control cells. This is the first description of cutaneous antibodies against SMRV in grass carp.

Development and characterization of monoclonal antibodies to spring viraemia of carp virus

Five monoclonal antibodies (mAbs) against spring viraemia of carp (SVCV0504, isolated from common carp in China) were produced from mice immunized with purified virus preparations. The virion of SVCV contains five structural proteins, representing the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (Q. Western blotting analysis revealed that three mAbs (1145, IE10, and 11-17) recognized specifically to a single protein of 47 kDa (N), the mAb 3G4 reacted with, two SVCV0504 proteins of 69 kDa (G) and 47 kDa (N), while the mAb 1A9 reacted with three SVCV0504 proteins of 69 kDa (G), 50 kDa (P), and 47 kDa (N). By indirect ELISA, two mAbs (1H5 and 11-17) showed cross-reactivity with pike fry rhabdovirus (PFRV), but no cross-reactions with the Siniperca chuatsi rhabdovirus (SCRV), Scophthalmus maximus rhabdovirus (SMRV), Paralichthys olivaceus rhabdovirus (PoRV) were demonstrated with the five mAbs. Indirect immunofluorescence showed intense fluorescence in the cytoplasm of the SVCV0504-infected epithelioma papulosum cyprini (EPC) cells in areas corresponding to the location of granular structures. The sucrose gradient-purified SVCV0504 particles could be detected successfully by these mAbs using immunodot blotting. mAb 1A9 could completely neutralize 100 TCID50 (50% tissue culture infective dose) of SVCV0504 at a dilution of 1:8. This is the first report of development of the neutralizing mAbs against SVCV. The mAb 1A9 was analyzed further and could be used to successfully detect viral antigens in the infected-EPC cell cultures or in cryosections from experimentally infected crucian carp (Carassius auratus) by immunohistochemistry assay. Furthermore, a flow cytometry procedure for the detection and quantification of cytoplasmic SVCV0504 in cell cultures was developed with mAb 1A9. At 28 h after inoculation with the virus (0.01 PFU/cell), 10.12% of infected cells could be distinguished from the uninfected cells. These mAbs will be useful in diagnostic test development and pathogenesis studies for fish rhabdovirus. (c) 2008 Elsevier B.V. All rights reserved.

Generation and characterization of monoclonal antibodies against the flounder Paralichthys olivaceus rhabdovirus

Two MAbs (3C7 and 3C9) against flounder Paralichthys olivaceus rhabdovirus (PORV) were generated with hybridoma cell fusion technology and characterized by an indirect enzyme-linked immunosorbent assay, isotype test, Western blot and immunodot analysis and immunofluorescence assay. Isotyping tests demonstrated that both of the two MAbs belonged to IgM subclass. Western blot analysis showed the MAbs reacted with 42, 30, and 22 kDa viral proteins, which were localized within the cytoplasm of PORV-infected grass carp ovary (GCO) cells analyzed by indirect immunofluorescences tests. The MAb 3C7 was also selected at random for detecting virus antigens in the inoculated grass carp tissues by immunohistochemistry assay. Flow cytometry tests showed that at the 36 h postinfection (0.25 PFU/cell), the 23% PORV-infected GCO cells could be distinguished from the uninfected cells with the MAb 3C7. Such MAbs could be useful for diagnosis and potential treatment of viral infection. (C) 2007 Elsevier B.V. All rights reserved.

基于RTI的车辆定位仿真系统数据传输研究

目前分布式体系结构的研究重点是提高系统的可扩展性、互操作性和可重用性，而对于实时性要求高的分布式仿真系统，还需要在HLA体系结构基础上，考虑如何提高系统的数据传输效率，以满足实时性要求。本文从应用层角度出发，从以下两个方面研究了改进策略： 一，从数据交互方面考虑，为了提高分布式仿真系统内部有效数据传输效率，满足系统实时性要求，以车辆定位仿真系统为问题原型，提出一种基于运行时间支撑系统数据分发管理（RTI-DDM）和套接字(Socket)的双层数据传输模型：一方面利用RTI-DDM来限定传输数据的范围，依据待交互的数据值域对数据的发送和接收进行过滤，有效降低系统内部冗余数据的传输；另一方面，利用Socket技术在仿真实体之间建立点对点的直接传输，从而提高系统的运行效率。对比实验结果表明，在相同的仿真交互数据量下，该模型相对于传统单层RTI数据传输模型，数据传输延时平均缩短70%。 二，从时间管理方面考虑，本文通过实验验证的方法，分析不同时间管理策略对仿真系统的数据传输和运行控制的影响，并针对车辆定位仿真系统中联邦成员之间的数据交互和逻辑控制关系特点，选择合适的时间管理策略，保证数据因果关系的正确性，进一步提高了系统的数据传输性能。 实验和应用结果表明，本文提出的改进策略简单有效，提高了系统数据传输效率，较好的解决了基于HLA/RTI的定位仿真系统的实时性问题。

卫星通信网络用户接入与路由选择技术研究

卫星移动通信作为下一代移动通信中一个必不可少的组成部分，用户接入切换和路由选择策略已经成为卫星移动通信领域的研究重点。同时对卫星通信网络建设方案及其关键技术进行充分的仿真建模论证也已经成为卫星通信系统建设前的一项必不可少的工作。本文围绕用户接入切换策略和路由选择策略开展了如下工作： 在卫星通信网络用户接入切换策略中，本文针对低轨卫星通信网络研究多星覆盖接入切换技术。首先在引入非均匀多业务模型的基础上，结合星上信道分配策略设计一种多业务组合加权接入策略。接着在基于HLA/RTI的卫星网络仿真系统之上进行用户接入仿真实现，并对最近卫星、最长覆盖时间和组合加权接入策略进行了仿真分析与比较，验证了组合加权策略下的系统性能。最后进一步仿真讨论加权系数取值不同对系统新呼叫阻塞概率、切换呼叫阻塞概率和切换请求到达概率的影响。 针对卫星通信网络路由策略，本文重点研究多层卫星网络，在建立星间链路预测模型的基础上，借鉴延时可容忍网络路由设计思想，设计一种链路中断容忍路由策略，利用非均匀时间段内卫星网络拓扑结构的可预测性进行路由表计算，同时提供动态的拥塞控制机制和基于洪范思想的故障中断容忍策略，解决由卫星运动、通信设备故障等引发链路中断情况下的路由问题。通过仿真，验证该路由策略的时空特性和链路利用率。

基于刚体运动学仿真的坦克训练模拟系统研究

根据坦克部队作战特点，以计算机技术、网络技术、系统仿真与模型方法为基础，将HLA与坦克分队战术训练相结合，把分散在不同地点的人与设备“连接”到同一模拟训练环境中；将虚拟现实技术与半实物仿真模拟器相结合，建立吴有时空一致性的系统合成的虚拟“战场环境”；将刚体运动学与解析几何相结合，解决坦克实体模型视景仿真和坦克直线运动与转向运动中的六自由度刚体运动学模拟技术。遵循这一技术路线，完成了以计算机仿真技术与军事训练专业的复合应用为目的的分布式坦克训练模拟与分析系统。