AIDS 免疫治疗相关腺病毒载体的构建

艾滋病（AIDS）是人类免疫缺陷病毒（HIV）感染后引起的一种严重危害人类健 康的致死性传染病。抗HIV 药物挽救和延长了很多HIV 感染者的生命，提高了其生活 质量，但是仍然不能治愈AIDS 和预防传播。最终有效控制HIV 传播和感染的方法可能 仍将依赖于HIV 疫苗的应用。HIV 感染对感染者以及社会造成的灾难性后果使得发展 一个有效的艾滋病疫苗变得尤为紧迫和重要。 负载HIV-1 抗原的DC 回输到HIV-1 感染者体内可以诱导产生较强的抗HIV-1 细 胞免疫反应，这种免疫反应理论上和临床试验都表明治疗AIDS 有效，而且对HARRT 治疗能够产生很好的协同作用。我们拟用感染了重组腺病毒的DC，回输到HIV-1 感染 者体内，期望可以较好地控制病毒复制和阻止感染。为此，本研究我们制备了重组腺病 毒vAd-gp140、vAd-tat 和vAd-gp140-tat，为后续研究奠定基础。 结构蛋白Env 是激发抗体反应的抗原，由于Env 全长有较大细胞毒性，本文采用 了截短的gp140 分子，删除了gp41 的胞内段，降低了gp140 蛋白的细胞毒性。同时保 留了gp41 的跨膜区，表达的蛋白可被正确地锚定在细胞表面，提高蛋白的免疫原性。 将gp140 分子克隆到复制缺陷型的腺病毒载体中，用Wester Blotting 方法检测到gp140 在293 细胞中的表达。 有效的抗 HIV-1 的疫苗应该能够同时激发针对多种亚型病毒株的细胞和体液免疫 反应。早期病毒蛋白激发的CTL 应答在控制病毒载量上更为有效，而且Tat 蛋白的重 要免疫抗原表位和功能区域在不同HIV-1 病毒株之间是高度保守的。Tat 蛋白的多种生 物学功能使得它成为较强的免疫原、共抗原和佐剂，激发T 细胞抗原表位的Th1 型反 应和CTL 反应，扩大体内T 细胞识别的抗原表位谱，提高T 细胞特异性免疫反应。本 文扩增了HIV-1ⅢB 的tat 基因，克隆到复制缺陷性的腺病毒中，构建了重组腺病毒 vAd-tat，并在293 细胞中表达了分子量大小为15kDa 的蛋白。早期蛋白和结构蛋白的联合免疫能够全面地控制病毒复制，在动物实验中一定程度 上保护了猴子。我们将已得到表达的gp140 和tat 基因进行融合表达。利用融合PCR 技 术，扩增gp140 和tat 的融合基因，构建携有HIV-1 gp140-tat 融合基因的重组腺病毒 vAd-gp140-tat。gp140-tat 在293 细胞中的融合表达还需要进一步验证。 下一步的工作是将构建好的重组腺病毒感染DC，检测外源基因在DC 中的表达水 平，对DC 表面分子表型的影响以及对DC 功能的影响。

猕猴MHC研究及在AIDS动物模型中的意义

主要组织相容性复合体(MHC)是与免疫应答和移植排斥直接相关的一组基因群.由于MHC高度的多态性,使其在脊椎动物的免疫遗传、进化、保护以及与疾病的相关性等方面的研究倍受关注.猕猴MHC尤其是MHC Ⅰ类基因的组成与人类有显著差异,一个单体型中存在多个Mamu-A和Mamu-B基因.在猕猴AIDS模型中,MHC对病毒的免疫逃逸以及对AIDS疫苗的研究均有十分重要的作用,某些MHC Ⅰ类和MHCⅡ类基因能够显著延缓猕猴AIDS疾病的进展,为在人体中理解MHCⅠ类等位基因与免疫保护和控制病毒复制的相关性提供了一个良好的模型.

人类AIDS 的研究替身———非人灵长类动物模型

　非人灵长类动物模型在艾滋病发病机理、传播途径、免疫反应以及疫苗开发药物治疗等方面的研究具有重要作 用。根据不同的免疫缺陷病毒感染不同的灵长类动物,可将这一模型分为不同的种类, 其中HIV/ 黑猩猩模型、SIV/ 猕猴模型和SHIV/ 猕猴模型是目前应用最为广泛的模型。这几种模型都各有其优缺点,本文将简单介绍这几种艾滋 病非人灵长类动物模型的研究概况。

树突状细胞在AIDS灵长类动物模型疾病进程中的作用

非人灵长类动物模型在艾滋病(AIDS)发病机制、传播途径、疫苗和药物等方面的研究中具有重要作用。树突状细胞(DC)作为最重要连接先天免疫与获得性免疫的抗原递呈细胞,在AIDS发病进程中扮演着重要的角色。研究非人灵长类AIDS动物模型中DC亚群数量、表型以及功能的变化,对揭示AIDS发病机制具有十分重要的意义。该文将重点总结近些年来DC亚群在AIDS动物模型发病机制中的作用研究进展,为以后的研究提供思路。

Uganda: fighting HIV and AIDS

African fisherfolk suffering from HIV are subject to stigma, denigration and disempowerment, all of which weaken their voice in civil society...

Issues of HIV/AIDS/ health among the fisher communities of Lake Albert and Kyoga

The fisheries resources of Lakes Albert and Kyoga present a high potential for economic growth, food, employment and foreign earnings. However, livelihoods appear to be compromised with the emergence and rapid spread of HIV/AIDS in the fisher communities of L. Albert and Kyoga. HIV/AIDS is considered a silent epidemic that is unique, posing a great challenge to the fisheries managers, health service providers, development planners and the resource users themselves. Fishers have high HIV prevalence, as well as AIDS-related illnesses and mortality rates. The high HIV prevalence rates among the fishing communities in Uganda is between 10-40% compared to the national rates which lie between 6% and 7%. This indicates that the national programmes have not adequately addressed the plight of the fishing communities of Lakes Albert, and Kyoga and the consequences have been devastating. Men and women living in fishing villages across the world have been found to be between five and ten times more vulnerable to the disease than other communities (Tarzan et al 2005, FAO, 2007). The present prevalence rates among the fishing communities stands at 10 to 40 % (LVFO, 2008). Meanwhile the same fishing communities are the essential labour for the Lakes’ fishery industry which is thriving nationally and internationally. That resource potentially can alleviate poverty and the HIV/AIDS threat. Fishing communities are the hidden victims of the disease, mixing patterns with the general population could act as a reservoir of infection that could spill over into the general population to drive the epidemic. On L. Albert, a quarter of the fisher folk were HIV-positive by 1992 compared to 4% in a nearby Agricultural village. Since then, there have been no targeted studies to address or monitor the prevalence rates eight years later, yet the multiplicity factor is high. HIV/AIDS can be linked to unsustainable fisheries, as the labour force available would not go to deep waters to fish, instead would fish in the shallow waters as a coping mechanism. A further effect is the loss to National and local economies and reduced nutritional security for the wider population. HIV/AIDS remains a significant challenge that has created a mosaic of complexity in the fishery sector. This needs to be addressed. It is, therefore, paramount that a comprehensive study was under taken to address this pandemic and the phenomenon of HIV/AIDS based on the study objectives. 1. To determine the trend in HIV/AIDS infection among fishing communities and the factors affecting it 2. To assess the impacts of HIV/AIDS on fish production and the implications for fisheries management.

Shortening the decision distance: Selecting decision aids for improved technology investment performance

Deciding which technology to invest in is a recurring issue for technology managers, and the ability to successfully identify the right technology can be a make or break decision for a company. The effects of globalisation have made this issue even more imperative. Not only do companies have to be competitive by global standards but increasingly they have to source technological capabilities from overseas as well. Technology managers already have a variety of decision aids to draw upon, including valuation tools, for example DCF and real options; decision trees; and technology roadmapping. However little theory exists on when, where, why or even how to best apply particular decision aids. Rather than developing further techniques, this paper reviews the relevance and limitations of existing techniques. This is drawn from an on going research project which seeks to support technology managers in selecting and applying existing decision aids and potentially in the design of future decision aids. It is intended that through improving the selection of decision aids, decision performance can be increased, leading to more effective allocation of resources and hence competitive advantage. (c) 2006 PICMET.

Biologically inspired visual odometer for navigation of a flying robot

Experimental research in biology has uncovered a number of different ways in which flying insects use cues derived from optical flow for navigational purposes, such as safe landing, obstacle avoidance and dead reckoning. In this study, we use a synthetic methodology to gain additional insights into the navigation behavior of bees. Specifically, we focus on the mechanisms of course stabilization behavior and visually mediated odometer by using a biological model of motion detector for the purpose of long-range goal-directed navigation in 3D environment. The performance tests of the proposed navigation method are conducted by using a blimp-type flying robot platform in uncontrolled indoor environments. The result shows that the proposed mechanism can be used for goal-directed navigation. Further analysis is also conducted in order to enhance the navigation performance of autonomous aerial vehicles. © 2003 Elsevier B.V. All rights reserved.

Pipeline Architecture and Parallel Computation-Based Real-Time Stereovision Tracking System for Surgical Navigation

This paper studies the development of a real-time stereovision system to track multiple infrared markers attached to a surgical instrument. Multiple stages of pipeline in field-programmable gate array (FPGA) are developed to recognize the targets in both left and right image planes and to give each target a unique label. The pipeline architecture includes a smoothing filter, an adaptive threshold module, a connected component labeling operation, and a centroid extraction process. A parallel distortion correction method is proposed and implemented in a dual-core DSP. A suitable kinematic model is established for the moving targets, and a novel set of parallel and interactive computation mechanisms is proposed to position and track the targets, which are carried out by a cross-computation method in a dual-core DSP. The proposed tracking system can track the 3-D coordinate, velocity, and acceleration of four infrared markers with a delay of 9.18 ms. Furthermore, it is capable of tracking a maximum of 110 infrared markers without frame dropping at a frame rate of 60 f/s. The accuracy of the proposed system can reach the scale of 0.37 mm RMS along the x- and y-directions and 0.45 mm RMS along the depth direction (the depth is from 0.8 to 0.45 m). The performance of the proposed system can meet the requirements of applications such as surgical navigation, which needs high real time and accuracy capability.

Pipeline Architecture and Parallel Computation-Based Real-Time Stereovision Tracking System for Surgical Navigation

This paper studies the development of a real-time stereovision system to track multiple infrared markers attached to a surgical instrument. Multiple stages of pipeline in field-programmable gate array (FPGA) are developed to recognize the targets in both left and right image planes and to give each target a unique label. The pipeline architecture includes a smoothing filter, an adaptive threshold module, a connected component labeling operation, and a centroid extraction process. A parallel distortion correction method is proposed and implemented in a dual-core DSP. A suitable kinematic model is established for the moving targets, and a novel set of parallel and interactive computation mechanisms is proposed to position and track the targets, which are carried out by a cross-computation method in a dual-core DSP. The proposed tracking system can track the 3-D coordinate, velocity, and acceleration of four infrared markers with a delay of 9.18 ms. Furthermore, it is capable of tracking a maximum of 110 infrared markers without frame dropping at a frame rate of 60 f/s. The accuracy of the proposed system can reach the scale of 0.37 mm RMS along the x- and y-directions and 0.45 mm RMS along the depth direction (the depth is from 0.8 to 0.45 m). The performance of the proposed system can meet the requirements of applications such as surgical navigation, which needs high real time and accuracy capability.

AIDS猕猴模型在HIV疫苗研究中的应用

：对HIV 疫苗的研究一直是国际上艾滋病方面研究的热点和难点。动物模型则为疫苗研究必不可缺少的 重要工具，缺乏合适的动物模型很大程度上制约了AIDS疫苗的研究。目前在国际上SIV 或SHIV 感染的猕猴模 型为最常用的AIDS研究模型，受猕猴背景及病毒特性等多种因素的影响，使得以上两种模型在HIV疫苗研究中 仍存在一定的局限性。为了更好地发挥猕猴模型在HIV疫苗研究中的巨大潜力，开发理想的AIDS猕猴模型已成 为目前HIV疫苗研究的首要任务。本文简要介绍了AIDS疫苗的研发策略、研发概况以及SIV／SHIV猕猴模型在 HIV疫苗中的应用，并对其中存在的问题及其应用前景进行了探讨。

Establishment of AIDS Animal Model with SIVmac239 Infected Chinese Rhesus Monkey

In the present research, two Chinese rhesus monkeys were inoculated intravenously with 5000 TCID50 of SIVmac239. The changes in the numbers of CD4+ T lymphocyte in peripheral blood, plasma viral loads, proviral DNA and humoral antibodies against virus were periodically monitored during 121 days. At the early stage of infection, proviral DNA had been detected in PBMCs, and infectious SIVmac239 virus had been isolated from PBMCs. At the same period, the numbers of CD4+ T lymphocytes were significantly decreased, and maintained at low level during the 121-day period of infection. Plasma viral loads reached the peak at week 2 post-inoculation and kept at a steady state subsequently. Moreover, antibodies against viral proteins were detected from plasma. All the results showed that the two Chinese rhesus monkeys had been infected with SIVmac239 successfully. This animal model can be applied for further AIDS researches.

TRIM5α基因的分子进化及AIDS 免疫治疗相关腺病毒载体的构建

本文包括两方面的研究工作。第一部分对TRIM5α基因在灵长类动物中的分 子进化进行了详尽的研究，希望能从分子进化的角度认识该基因在灵长类动物抵 抗病毒感染尤其是在限制HIV-1 感染过程中所起的作用，并希望能够从中寻找适 合用于AIDS 基因治疗的基因。第二部分构建了艾滋病治疗性DC 疫苗相关的腺 病毒载体，包括表达HIV-1 gp120 基因、表达HIV-1 gp41 基因、表达HSP70 基 因以及仅表达绿色荧光蛋白基因的重组腺病毒，为利用HIV-1 抗原刺激的DC 免 疫治疗AIDS 的研究奠定了基础。 在长期的HIV-1 和AIDS 研究中，人们发现很多种旧大陆猴不能被HIV-1 感 染，而旧大陆猴中的平顶猴却对HIV-1 有易感性，并且对SIV 的感染也表现出较 其它旧大陆猴更为严重的症状，TRIM5α基因的发现解释了这一“历史之谜”。人 们发现旧大陆猴的TRIM5α基因具有很强的抗HIV-1 的功能，是旧大陆猴抵抗 HIV-1 感染必须的免疫因子；并且进一步证明TRIM5α是灵长类动物中普遍存在 的、具有抗多种逆转录病毒功能的天然免疫因子。我们通过对TRIM5α基因在灵 长类动物中的分子进化研究发现，证明该基因在进化过程中表现出很强的达尔文 正向选择作用，尤其是其负责识别逆转录病毒衣壳蛋白的SPRY 结构域。TRIM5 α基因表现出的正向选择很可能是灵长类动物在长期的进化过程中反复被病毒 感染的选择压力造成的，这也从另一个方面证明TRIM5α基因是一个普遍存在的、 具有抗病毒功能的天然免疫因子。对不同灵长类动物的TRIM5α基因序列分析发 现，该基因的序列变化很大，这可能是不同灵长类动物被HIV-1 感染后表现不同 症状的原因；基于密码子的中性检测证明，TRIM5α基因SPRY 结构域上第347、 354、552 位氨基酸表现出很强的正向选择，说明这些位点可能与其抗病毒功能 关系紧密。另外，我们发现平顶猴的TRIM5α基因与CypA 基因以一种新的方式 融合，这种融合方式不同于鹰猴中该基因与CypA 基因的融合，而且与具有较强抗HIV-1 功能的恒河猴的TRIM5α基因比较，平顶猴的TRIM5α基因在几个与其 抗病毒功能紧密相关的位点上的氨基酸也发生了变化，这可能是其被HIV-1 和 SIV 感染后表现出较其它旧大陆猴更为严重症状的原因。 将经过HIV-1 抗原刺激的DC 回输到HIV-1 感染者体内可以诱导产生较强的 抗HIV-1 细胞免疫反应，这种免疫反应理论上可以治疗AIDS，而且对HARRT 治 疗能够产生很好的协同作用；HSP70 蛋白可以提高DC 对抗原的加工和呈递。为 了检测经HIV-1 抗原刺激的DC 免疫治疗AIDS 的实际效果，同时检验通过HSP70 增强DC 对HIV-1 抗原的加工和呈递功能，发现并解决这一治疗方案存在的问题， 我们首先从HIV-1 的基因组及带有HSP70 基因的质粒上克隆了gp120、gp41 和 HSP70 基因，测序及序列分析证明所克隆的基因没有发生影响表达的突变。然后 我们分别构建了表达有关基因的重组腺病毒载体。在荧光显微镜下可以观察到感 染后的细胞内绿色荧光蛋白报告基因的表达，证明成功构建了四种重组腺病毒， 而PCR 扩增也证明在其中三种重组腺病毒的基因组上确实分别带有gp120、gp41、 HSP70 基因。同时我们还构建了不表达外源基因的重组腺病毒，作为未来实验中 的对照载体。未来的研究工作包括目的基因表达检测，重组腺病毒感染DC 及腺 病毒感染DC 功能分析等。

1、喜树碱类衍生物抗HIV构效关系与作用机制研究2、HIV/AIDS患者疱疹病毒感染状况及性病患者的HIV感染状况分析

1、喜树碱类衍生物抗HIV构效关系与作用机制研究 喜树碱为传统的抗肿瘤药物。本研究对经过化学结构修饰的喜树碱类衍生物进行抗HIV活性及作用机制的研究，并初步探讨了其抗HIV构效关系。 我们对喜树碱类衍生物A系列化合物A1(喜树碱)、A2(10-羟基喜树碱)及A3(7-羟基喜树碱)进行了抗HIV活性检测。化合物A1和A3有较好的抗HIV-1和抗HIV-2活性，化合物A2没有显示抗HIV活性。表明化合物A1的C-10位上-OH基团修饰可能会降低抗HIV活性，化合物A1的C-7位上-CH2OH基团修饰和C-20位-CH3缺失可能会提高其抗HIV活性。对化合物A3和A1的抗HIV机制研究发现：二者对整合酶有一定的结合活性，对慢性感染H9/HIV-1ⅢB 和Jurkat/HIV-1ⅢB细胞中病毒复制没有抑制活性、不能阻断H9/HIV-1ⅢB与正常细胞间的融合，对重组的HIV-1蛋白酶和逆转录酶没有抑制活性。化合物A1和A3不具有选择性杀伤HIV-1ⅢB慢性感染的H9和Jurkat细胞系的作用。进一步进行化合物A3诱导 H9和H9/HIV-1ⅢB、Jurkat和Jurkat/HIV-1ⅢB的凋亡实验显示，化合物A3诱导感染HIV-1ⅢB和未感染病毒细胞的凋亡没有选择性。据此我们初步认为化合物A3和A1的抗HIV作用可能与抑制整合酶活性有关，该化合物可能还作用于其它靶点。 喜树碱类衍生物B系列中化合物B1为20(S)-O - [-O-( 1'-氧基-2',2',6',6'-四甲基哌啶-4'-丁二酸)]-20-喜树碱酯，化合物B2为20(S)-O - [-N-( 1'-氧基-2',2',6',6'-四甲基-1',2',5',6'-四氢吡啶酰胺)-4'-丙氨酸)]-20-喜树碱酯)。我们对化合物B1和B2进行了抗HIV活性检测。结果显示：化合物B2有较好的抗HIV-1和抗HIV-21、喜树碱类衍生物抗HIV构效关系与作用机制研究 喜树碱为传统的抗肿瘤药物。本研究对经过化学结构修饰的喜树碱类衍生物进行抗HIV活性及作用机制的研究，并初步探讨了其抗HIV构效关系。 我们对喜树碱类衍生物A系列化合物A1(喜树碱)、A2(10-羟基喜树碱)及A3(7-羟基喜树碱)进行了抗HIV活性检测。化合物A1和A3有较好的抗HIV-1和抗HIV-2活性，化合物A2没有显示抗HIV活性。表明化合物A1的C-10位上-OH基团修饰可能会降低抗HIV活性，化合物A1的C-7位上-CH2OH基团修饰和C-20位-CH3缺失可能会提高其抗HIV活性。对化合物A3和A1的抗HIV机制研究发现：二者对整合酶有一定的结合活性，对慢性感染H9/HIV-1ⅢB 和Jurkat/HIV-1ⅢB细胞中病毒复制没有抑制活性、不能阻断H9/HIV-1ⅢB与正常细胞间的融合，对重组的HIV-1蛋白酶和逆转录酶没有抑制活性。化合物A1和A3不具有选择性杀伤HIV-1ⅢB慢性感染的H9和Jurkat细胞系的作用。进一步进行化合物A3诱导 H9和H9/HIV-1ⅢB、Jurkat和Jurkat/HIV-1ⅢB的凋亡实验显示，化合物A3诱导感染HIV-1ⅢB和未感染病毒细胞的凋亡没有选择性。据此我们初步认为化合物A3和A1的抗HIV作用可能与抑制整合酶活性有关，该化合物可能还作用于其它靶点。 喜树碱类衍生物B系列中化合物B1为20(S)-O - [-O-( 1'-氧基-2',2',6',6'-四甲基哌啶-4'-丁二酸)]-20-喜树碱酯，化合物B2为20(S)-O - [-N-( 1'-氧基-2',2',6',6'-四甲基-1',2',5',6'-四氢吡啶酰胺)-4'-丙氨酸)]-20-喜树碱酯)。我们对化合物B1和B2进行了抗HIV活性检测。结果显示：化合物B2有较好的抗HIV-1和抗HIV-2活性，而化合物B1的抗HIV活性差。表明化合物B1的C-4’位-CH2被-NH取代，同时C-3’位-CH3修饰可能会提高其抗HIV活性。对化合物B2的抗HIV机制研究发现，化合物B2对慢性感染H9/HIV-1ⅢB细胞中病毒复制没有抑制活性、不能阻断H9/HIV-1ⅢB与正常细胞间的融合，对HIV-1蛋白酶、重组的HIV-1逆转录酶及整合酶没有抑制活性。化合物B2不具有选择性杀伤HIV-1ⅢB慢性感染的H9细胞系的作用。化合物B2抗HIV的作用机制还需进一步研究。 2、HIV/AIDS患者疱疹病毒感染状况及性病患者的HIV感染状况分析 疱疹病毒是AIDS患者合并感染的常见病原体。引起人类疾病的8种疱疹病毒与HIV感染及AIDS进展、机会性感染、恶性肿瘤密切相关。为了解HIV/AIDS患者人类8型疱疹病毒感染状况，我们检测了30例AIDS患者、40例HIV携带者及70例正常对照的液标本中8型疱疹病毒感染状况。采用ELISA法检测单纯疱疹病毒1型(HSV-1)、单纯疱疹病毒2型(HSV-2)、水痘-带状疱疹病毒(VZV)和巨细胞病毒(CMV)；采用PCR法检测EB病毒(EBV)、疱疹病毒6型(HHV-6)、疱疹病毒7型(HHV-7)及疱疹病毒8型(HHV-8)。结果显示，HIV/AIDS患者中HSV-1、HSV-2、VZV、CMV、HHV-6、HHV-8 阳性率均高于健康体检者，其中AIDS患者VZV感染率与HIV携带者有显著性差异；在AIDS患者中多种疱疹病毒共感染普遍存在，必须重视HIV/AIDS患者合并疱疹病毒感染的防治。 性病可促进HIV的传播，了解性病患者的HIV感染状况及临床特征具有重要的意义。在自愿接受HIV咨询检测的基础上，对临床确诊的412例性病患者进行HIV-1/2抗体检测，并对其临床特征进行分析研究。结果显示412例性病患者的HIV检出率为2.9%。性病患者中检出HIV阳性率依次为：尖锐湿疣(6.2%)、生殖器疱疹(4.2%)、梅毒(3.4%)、淋病(1.5%)及非淋菌性尿道炎(1.0%)。83.3%合并感染HIV的性病患者存在多性伴，商业性行为普遍存在，安全套使用率极低现象。感染HIV的尖锐湿疣及生殖器疱疹患者以频繁复发为突出表现，1例合并感染HIV的梅毒患者半年即进展为神经梅毒。性病患者是HIV感染的重要高危人群，危险性行为是其感染HIV和其它性病的主要原因，应该加强性病患者的HIV检测。对临床上频繁复发的尖锐湿疣及生殖器疱疹患者、快速进展的梅毒患者应高度怀疑合并HIV感染的可能。