【作者】 阴慧娟；

【导师】 李迎新；

【作者基本信息】 天津医科大学 ， 生物医学工程， 2010， 博士

【摘要】 目的：艾滋病(Acquired immunodeficiency syndrome, AIDS)在全球已呈爆发性流行趋势,它严重威胁着人类的健康和全球经济的发展。由于缺乏有效的疫苗且临床现有抗艾滋病药物存在价格昂贵、依从性差、易产生耐药性以及不能清除病毒等缺点,探索新的有效的抗艾滋病方法仍是当前研究的热点。光动力疗法(Photodynamic Therapy, PDT)是一种激光医学领域的新方法。早在上世纪90年代,作为血液制品净化的手段,PDT灭活人免疫缺陷病毒(Human Immunodeficiency Virus, HIV)病毒已经成为研究焦点,但是由于受光敏剂副作用以及激光器制造技术落后所限,该研究发展缓慢。近年来新型光敏剂不断问世,激光技术日新月异,为PDT用于艾滋病防治的研究提供了条件。本文将通过研究新型光敏剂血卟啉单甲醚(hematoporphyrin monomethyl ether, HMME)诱导的PDT对HIV感染的抑制作用,探索PDT防治艾滋病的可能和途径。以HIV-1ⅢB病毒株为代表研究HMME-PDT对HIV病毒复制各个阶段的抑制作用,探索PDT是否具有抗HIV活性及其机制；分析光敏剂、光以及氧对PDT抗HIV活性效应的影响；检测PDT对HIV野生株、耐药株以及各亚型病毒株的抑制效应,评价PDT抗HIV的临床前药效学效应,为PDT在临床上治疗艾滋病提供理论基础和实验依据。方法：1.检测PDT对HIV-1ⅢB急性感染MT4细胞死亡的保护,对HIV-1ⅢB慢性感染H9细胞中P24抗原的抑制,对C8166与HIV-1ⅢB慢性感染H9细胞融合和游离HIV-1ⅢB病毒与C8166细胞融合的抑制,以及对HIV-1ⅢB游离病毒的灭活,探讨PDT的抗HIV活性效应及机制；2.检测不同浓度HMME以及不同功率密度和能量密度下PDT对游离HIV-1ⅢB游离病毒的灭活作用,检测两种抗氧化剂叠氮钠和甘露醇作用下PDT对游离HIV-1ⅢB游离病毒的灭活作用,分析PDT抗HIV活性的影响因素：3.检测PDT对以HIV-1ⅢBA17、HIV-1 L10R/M46I/L63P/V82T/I84V、HIV-1 Protease Gene Mutants RF/V82F/184V、pNL4-3 gp41 (36G) N42S、pNL4-3 gp41 (36G) V38A, N42T为代表的耐药株、以HIV-1KM018和HIV-1SF162为代表的野生株、以及以HIV-2CBL20和HIV-2ROD为代表的2型HIV病毒株的灭活作用,评价PDT抗HIV的临床前药效学效应。结果：1. HMME-PDT能显著抑制HIV诱导的细胞-细胞融合(抑制率64.68%,P<0.05)和病毒-细胞融合(抑制率85%,P<0.05),并对游离病毒有很强的杀伤作用,最高可达到100%(P<0.05)。光动力疗法不能抑制HIV慢性感染H9/HIV-1ⅢB细胞产生P24抗原,不能保护HIV急性感染MT-4细胞的死亡(P>0.05)。2.在一定范围内,HMME-PDT灭活HIV-1ⅢB的效应与光敏剂浓度和能量密度呈正相关,而与功率密度呈反相关；两种抗氧化剂均能抑制HMME-PDT抗HIV-cell膜融合活性,甘露醇的抑制效应大于叠氮钠(P<0.05)。3. HMME-PDT对游离HIV-1ⅢBA17、HIV-1 L10R/M46I/L63P/V82T/I84V、HIV-1 Protease Gene Mutants RF/V82F/184V、pNL4-3 gp41 (36G) N42S、pNL4-3 gp41 (36G) V38A, N42T、HIV-1KM018、HIV-1SF162、HIV-2CBL20和HIV-2ROD等病毒株均有显著的灭活效应,抑制率可高达100%(P<0.05)。结论：HMME-PDT可显著抑制游离HIV-1ⅢB的感染和HIV-1ⅢB诱导的膜融合作用,其灭活游离HIV的效应与光敏剂浓度、激光能量密度、激光功率密度以及抗氧化剂密切相关。HMME-PDT对游离HIV野生株、耐药株和HIV-2亚型病毒株等有限制的灭活效应,显示其临床前药效学效应良好。这些研究结果提示HMME-PDT可能成为治疗艾滋病的一种新方法。更多还原

【Abstract】 Acquired immunodeficiency syndrome (AIDS) has spread worldwide currently, which is threatening human health and the development of world economics seriously. Photodynamic therapy (PDT) is a new method in laser medcine. In the 1990s, the study of photoinactivation of human immunodeficiency virus(HIV) in blood products was a focus of intense investigation but the side effects of photosensitizers and technical issues with lasers hampered research. Since these initial studies, a series of new photosensitizers have been developed and new laser technologies have emerged, making the prospect of treating AIDS by PDT more promising. The current study explored the potential of in vitro photoinactivation of HIV by PDT with a new photosensitizer, hematoporphyrin monomethyl ether (HMME).Objective:Through exploring the potential and mechanism of anti-HIV activities based on HMME-PDT, analysising the relationships between the anti-HIV effects of PDT and photosensitizers concentration, power density, energy density and antioxidants, evaluating the preclinical pharmacodynamic effects of PDT anti-HIV, we expected these studies could provide academic and experimental evidence for the treatment of AIDS using PDT in clinic.Methods:1. The protection assay in acute infected MT-4 was assessed by MTT method, reduction of p24 antigen expression level in chronically infected H9 was measured by ELISA, the inhibition of cell-cell and virus-cell fusion induced by HIV-1ⅢB were observed and the activities to inactivate HIV-1ⅢB free particles were detected by ELISA.2. The anti-HIV effects of PDT with different doses of photosensitizers, power density, and energy density, and in present of two antioxidants including sodium azide and D-mannitol were accessed by ELISA to explore the influential factors of PDT anti-HIV. 3. The activities to inhibit the infectivity of HIV free particles including HIV-1ⅢBA17、HIV-1 L10R/M46I/L63P/V82T/I84V、HIV-1 Protease Gene Mutants RF/V82F/184V、pNL4-3 gp41 (36G) N42S、pNL4-3 gp41 (36G) V38A, N42T、HIV-1KM018、HIV-1SF162、HIV-2CBL20 and HIV-2ROD were measured by ELISA to evaluate the preclinical pharmacodynamic effects of PDT anti-HIV.Results:1. HMME-PDT inhibited the cell-cell membrane fusion up to 64.68%(P<0.05) and virus-cell fusion up to 85%(P<0.05) significantly. Cell-free virus inhibition of up to 100%(P<0.05) was achieved by each treatment. However, PDT had little effect on the acute infection or chronic infection of HIV-1ⅢB.2. Within a certain range, the inhibition rates of cell-free HIV-1ⅢB were typically increased in proportion to the augment of HMME concentration and energy density, but in inverse proportion to power density. Both sodium azide and D-mannitol could weaken the anti-HIV-cell fusion activities of PDT, and the latter done better (P<0.05).3. The infectivity of cell-free virus HIV-1ⅢBA17、HIV-1 L10R/M46I/L63P/V82T/I84V、HIV-1 Protease Gene Mutants RF/V82F/184V、pNL4-3 gp41 (36G)N42S、pNL4-3 gp41 (36G) V38A, N42T、HIV-1KM018、HIV-1SF162、HIV-2CBL20 and HIV-2ROD were all been inhibited significantly by HMME-PDT, with the inhibition rate up to 100%(P<0.05).Conclusion:HMME-PDT could inhibit the activity of HIV-induced membrane fusion and the infectivity of cell-free HIV. The anti-HIV effects of HMME-PDT are associated with HMME concentration, energy density, power density and antioxidant. These results suggest that PDT may be a promising new treatment for HIV infected individuals, especially those with high viral load.更多还原