杨梅素拮抗SEVI介导的HIV-1感染增强作用及其机制研究
发布时间：2018-09-06 16:17
【摘要】：研究目的:杀微生物剂(Microbicides)是一类含有抗HIV成分的凝胶、乳脂、栓剂、药膜或海绵,一般在性交前置入使用者的阴道或肛门内,用于预防HIV和其它性传播病原体的传播。由于其是一种可被女性控制的有效避免HIV感染的手段,因此成为预防艾滋病的一种新选择。但遗憾的是,迄今为止尚无杀微生物剂类药物问世。让人费解的是,多聚阴离子类杀微生物剂在临床前动物实验中均显示较好的预防HIV效果,但在临床试验阶段中却屡屡失败。导致杀微生物剂临床试验失败的原因是什么呢?由于动物攻毒过程中一般都直接采用PBS稀释病毒,而杀微生物剂用药部位多为阴道或直肠,不能忽视精液对杀微生物剂药效的影响。有研究表明,精液中的前列腺酸性磷酸酶(prostatic acidic phosphatase,PAP)的降解多肽片段PAP248-286,可以形成淀粉样纤维,并显著增强HIV感染,被命名为精液源性病毒感染增强因子(semen-derived enhancer of viral infection,SEVI)。随后,来源于精囊凝固蛋白的多肽片段SEM1和SEM2,以及同样来自酸性磷酸酶蛋白的N末端多肽片段PAP85-120先后被报道。上述这些精液源性的多肽片段均能形成淀粉样纤维,并增强HIV感染,极有可能是导致多聚阴离子类杀微生物剂临床试验失败的主要原因。据此,精液源性的淀粉样纤维成为抗HIV感染药物研发的新靶点。如果利用药物阻止精液源性淀粉样纤维的形成或阻断HIV与纤维结合,理论上可降低淀粉样纤维对病毒的感染增强作用,从而降低HIV的感染率。其中,SEVI为最早发现的精液源性淀粉样纤维,其结构稳定,在精液中含量较多,研究也最为深入,因此本文拟将SEVI作为主要的药物作用靶点进行研究。由于杀微生物剂必须具备高效、安全、产业化生产以及成本低等特性,而我国中草药天然资源众多,价格低廉,主要用于药品、保健食品、烟草、化妆品的原料或辅料等。其中,杨梅科杨梅属植物杨梅的黄酮类有效成分杨梅素(Myricetin,Myr)被报道对Aβ,Tau蛋白等多种淀粉样纤维的形成具有抑制作用。因此,我们推测杨梅素对精液源性淀粉样纤维的形成也具有同样的抑制作用。本项目以SEVI为药物作用靶点,通过一系列生物物理及生物化学手段评估杨梅素对SEVI淀粉样纤维形成及其功能的影响,深入探讨杨梅素拮抗SEVI介导的HIV-1感染增强作用及作用机制。同时,杨梅素本身具有较强的抗HIV-1病毒活性。因此,杨梅素极有可能被开发成为一种既具有高效抗HIV-1活性又可拮抗SEVI介导的HIV-1感染增强作用的双功能候选杀微生物剂,具有较大的临床应用价值。研究方法:1.杨梅素对SEVI淀粉样纤维形成的影响及其作用机制1)采用硫黄素T荧光法、刚果红染色法及透射电镜法检测杨梅素对SEVI淀粉样纤维形成的作用。2)采用圆二色谱法检测杨梅素对SEVI二级结构β-sheet形成的影响。3)选用HIV-1感染克隆株及TZM-b1细胞感染模型,检测经杨梅素处理后形成的SEVI对HIV-1病毒的感染增强作用。4)采用免疫印迹法和光催化酶联反应,研究杨梅素对SEVI形成阶段的影响。5)采用计算机模拟分子对接法,预测杨梅素与SEVI的相互作用位点。2.杨梅素对SEVI介导HIV-1感染增强作用的影响1)采用流式细胞术,免疫荧光显微镜观察杨梅素对SEVI介导病毒感染增强作用的影响。2)Virus 及Zeta-potential电势的方法研究杨梅素对SEVI与HIV-1病毒颗粒吸附作用的影响。3.杨梅素对精液中淀粉样纤维的形成及病毒感染增强作用的影响1)采用硫黄素T荧光法检测杨梅素对精液中淀粉样纤维形成的影响。2)采用TZM-b1细胞感染模型检测杨梅素对精液增强病毒感染能力的影响。3)检测精液环境中杨梅素与各类ARVs药物协同抗HIV-1作用。4.杨梅素对已形成的成熟SEVI淀粉样纤维的降解作用1)采用硫黄素T荧光法、刚果红染色及透射电镜法检测杨梅素对成熟的SEVI淀粉样纤维的降解作用。2)采用圆二色谱法检测杨梅素对成熟的SEVI淀粉样纤维二级结构β-sheet的影响。3)采用TZM-b1细胞感染模型检测杨梅素对降解后SEVI增强病毒感染能力的影响。研究结果:1.杨梅素能剂量依赖性地抑制SEVI淀粉样纤维的形成,75 μg/ml的杨梅素在48 h内能完全抑制淀粉样纤维的形成。圆二色谱结果表明,杨梅素能显著抑制淀粉样纤维的典型二级结构β-sheet片层的形成。对其抑制作用环节的研究发现,杨梅素可抑制SEVI早期寡聚物形成和纤维延长的全过程。计算机模拟分子对接发现,杨梅素可通过共价结合作用,与PAP248-286多肽的Leu-258、Gln-259、Val-264、Leu-268、Met-271 及 Arg-273 等位点特异性结合。2.杨梅素通过降低SEVI表面正电势,抑制SEVI对HIV-1病毒颗粒的吸附作用,从而拮抗SEVI介导的HIV-1感染增强作用。3.杨梅素能剂量依赖性地抑制精液中淀粉样纤维的形成,5 μg/ml的杨梅素可特异性阻断精液所介导的HIV-1感染增强作用。在精液存在下,杨梅素能与多种抗逆转录病毒药物,如 zidovudine(AZT)、raltegravir(RAL),maraviroc(MAR),tenofovir(TNF),nevirapine(NVP),efavirenz(EFV)等,产生协同抗 HIV-1 活性,其拮抗系数CI指数均小于0.5。4.杨梅素可迅速降解已经形成的成熟SEVI淀粉样纤维,从而阻断SEVI介导的HIV-1病毒感染增强作用。结论:杨梅素能显著抑制SEVI早期寡聚物形成和纤维延长的全过程,抑制SEVI对HIV-1病毒颗粒的吸附作用拮抗SEVI介导的HIV-1感染增强作用。此外,杨梅素可抑制精液介导的HIV-1感染增强作用,并能在精液存在下与多种ARVs药物发生协同抗HIV-1作用。同时,杨梅素也可以降解成熟的SEVI淀粉样纤维,从而破坏SEVI介导的HIV-1病毒感染增强作用。由于杨梅素本身也具有抗HIV-1病毒活性,因此,杨梅素极有可能被开发成为一种既具有高效抗HIV-1活性又可拮抗SEVI介导的HIV-1感染增强作用的双功能候选杀微生物剂,具有较大的临床应用价值。
[Abstract]:OBJECTIVE: Microbicides are gels, creams, suppositories, membranes, or sponges containing anti-HIV ingredients, usually placed in the vagina or anus of the user before intercourse to prevent the spread of HIV and other sexually transmitted pathogens. Because they are a woman-controlled and effective means of preventing HIV infection, they have become Unfortunately, no microbicides have been developed so far. It is difficult to understand that polyanionic microbicides have been shown to be effective in the prevention of HIV in preclinical animal experiments, but failed repeatedly in clinical trials, leading to the failure of microbicides clinical trials. Because PBS is usually used to dilute the virus directly in the course of viral attack, and most microbicides are used in vagina or rectum, the effect of semen on the efficacy of microbicides can not be ignored. 8-286, which can form amyloid fibers and significantly enhance HIV infection, was named semen-derived enhancer of viral infection (SEVI). Subsequently, polypeptide fragments from seminal vesicle coagulation protein, SEM1 and SEM2, and also from acidic phosphatase protein, N-terminal polypeptide fragment PAP85-120, were reported. These semen-derived polypeptide fragments can form amyloid fibers and enhance HIV infection, which is likely to be the main reason for the failure of clinical trials of polyanionic microbicides. The formation of powdery fibers or blocking the binding of HIV and fibers can theoretically reduce the enhancement of amyloid fibers to the infection of the virus, thereby reducing the infection rate of HIV. Among them, SEVI is the earliest discovered semen-derived amyloid fibers, which has stable structure, more content in semen and is the most in-depth study. Therefore, this paper intends to take SEVI as the main one. The target of drug action is studied. Because microbicides must have the characteristics of high efficiency, safety, industrial production and low cost, Chinese herbal medicine has many natural resources and low price, which are mainly used in medicine, health food, tobacco, cosmetics and other raw materials or accessories. Myricetin (Myr) has been reported to inhibit the formation of amyloid fibers such as Abeta and Tau proteins. Therefore, we speculate that myricetin also inhibits the formation of semen-derived amyloid fibers. The effect of myricetin on the formation and function of SEVI amyloid fibers and the mechanism of myricetin antagonizing SEVI-mediated HIV-1 infection were investigated. Myricetin itself has a strong anti-HIV-1 activity. Therefore, myricetin may be developed as an effective anti-HIV-1 activity and anti-SEVI-mediated HIV-1 antagonist. Methods: 1. Effect of myricetin on SEVI amyloid fiber formation and its mechanism 1) Thioflavin T fluorescence, Congo red staining and transmission electron microscopy were used to detect the effect of myricetin on SEVI amyloid fiber formation. 2) Circular dichroism was used. The effect of myricetin on the formation of SEVI secondary structure beta-sheet was detected by spectroscopy. 3) HIV-1 infection clone and TZM-b1 cell infection model were used to detect the enhanced effect of SEVI on HIV-1 infection. 4) Immunoblotting and photocatalytic enzyme-linked reaction were used to study the effect of myricetin on the formation of SEVI. The interaction site between myricetin and SEVI was predicted by computer-simulated molecular docking method. 2. The effect of myricetin on SEVI-mediated HIV-1 infection enhancement 1) The effect of myricetin on SEVI-mediated virus infection enhancement was observed by flow cytometry and immunofluorescence microscopy. 2) Virus and Zeta-potential potential potential were used to study the effect of myricetin on SEVI. Effects of myricetin on the formation of amyloid fibers in semen and the enhancement of viral infection 1) Effects of myricetin on amyloid fibers formation in semen were detected by Thioflavin T fluorescence assay. 2) Effects of myricetin on the enhancement of viral infection in semen were detected by TZM-b1 cell infection model. 3) Detecting the synergistic anti-HIV-1 effect of myricetin and various ARVs drugs in semen environment. 4. Degradation of mature SEVI amyloid fibers by myricetin 1) Degradation of mature SEVI amyloid fibers by Thioflavin T fluorescence, Congo red staining and transmission electron microscopy. 2) Detection of myricetin on mature SEVI amyloid fibers by circular dichroism The effect of myricetin on the secondary structure of SEVI amyloid fibers was investigated by TZM-b1 cell infection model. The results showed that myricetin could inhibit the formation of SEVI amyloid fibers in a dose-dependent manner and 75 ug/ml myricetin could completely inhibit the formation of SEVI amyloid fibers in 48 h. The results of circular dichroism showed that myricetin could significantly inhibit the formation of typical secondary structure beta-sheet lamellae of amyloid fibers. The inhibition mechanism of myricetin was found to inhibit the formation of early SEVI oligomers and the whole process of fiber elongation. Myricetin can inhibit SEVI-mediated HIV-1 infection by reducing the positive potential of SEVI and inhibiting the adsorption of SEVI to HIV-1 particles. 3. Myricetin can inhibit the enhancement of SEVI-mediated HIV-1 infection in a dose-dependent manner. In the presence of semen, myricetin can produce synergistic anti-HIV-1 activity with a variety of antiretroviral drugs, such as zidovudine (AZT), raltegravir (RAL), maraviroc (MAR), tenofovir (TNF), nevirapine (NVP), efavirenz (EFV), etc. The CI index was less than 0.5.4. Myricetin could rapidly degrade the mature SEVI amyloid fibers, thus blocking the SEVI-mediated enhancement of HIV-1 infection. Conclusion: Myricetin could significantly inhibit the whole process of oligomer formation and fiber elongation in early SEVI, and inhibit the adsorption of SEVI on HIV-1 virus particles. In addition, myricetin can inhibit the enhancement of semen-mediated HIV-1 infection and synergistic anti-HIV-1 effect with a variety of ARVs drugs in the presence of semen. Myricetin can also degrade mature SEVI amyloid fibers, thereby destroying SEVI-mediated HIV-1 infection enhancement. Myricetin has anti-HIV-1 activity, so it is very likely to be developed as a bifunctional candidate microbicide with high anti-HIV-1 activity and anti-SEVI-mediated HIV-1 infection enhancement.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96
本文编号：2226883
[Abstract]:OBJECTIVE: Microbicides are gels, creams, suppositories, membranes, or sponges containing anti-HIV ingredients, usually placed in the vagina or anus of the user before intercourse to prevent the spread of HIV and other sexually transmitted pathogens. Because they are a woman-controlled and effective means of preventing HIV infection, they have become Unfortunately, no microbicides have been developed so far. It is difficult to understand that polyanionic microbicides have been shown to be effective in the prevention of HIV in preclinical animal experiments, but failed repeatedly in clinical trials, leading to the failure of microbicides clinical trials. Because PBS is usually used to dilute the virus directly in the course of viral attack, and most microbicides are used in vagina or rectum, the effect of semen on the efficacy of microbicides can not be ignored. 8-286, which can form amyloid fibers and significantly enhance HIV infection, was named semen-derived enhancer of viral infection (SEVI). Subsequently, polypeptide fragments from seminal vesicle coagulation protein, SEM1 and SEM2, and also from acidic phosphatase protein, N-terminal polypeptide fragment PAP85-120, were reported. These semen-derived polypeptide fragments can form amyloid fibers and enhance HIV infection, which is likely to be the main reason for the failure of clinical trials of polyanionic microbicides. The formation of powdery fibers or blocking the binding of HIV and fibers can theoretically reduce the enhancement of amyloid fibers to the infection of the virus, thereby reducing the infection rate of HIV. Among them, SEVI is the earliest discovered semen-derived amyloid fibers, which has stable structure, more content in semen and is the most in-depth study. Therefore, this paper intends to take SEVI as the main one. The target of drug action is studied. Because microbicides must have the characteristics of high efficiency, safety, industrial production and low cost, Chinese herbal medicine has many natural resources and low price, which are mainly used in medicine, health food, tobacco, cosmetics and other raw materials or accessories. Myricetin (Myr) has been reported to inhibit the formation of amyloid fibers such as Abeta and Tau proteins. Therefore, we speculate that myricetin also inhibits the formation of semen-derived amyloid fibers. The effect of myricetin on the formation and function of SEVI amyloid fibers and the mechanism of myricetin antagonizing SEVI-mediated HIV-1 infection were investigated. Myricetin itself has a strong anti-HIV-1 activity. Therefore, myricetin may be developed as an effective anti-HIV-1 activity and anti-SEVI-mediated HIV-1 antagonist. Methods: 1. Effect of myricetin on SEVI amyloid fiber formation and its mechanism 1) Thioflavin T fluorescence, Congo red staining and transmission electron microscopy were used to detect the effect of myricetin on SEVI amyloid fiber formation. 2) Circular dichroism was used. The effect of myricetin on the formation of SEVI secondary structure beta-sheet was detected by spectroscopy. 3) HIV-1 infection clone and TZM-b1 cell infection model were used to detect the enhanced effect of SEVI on HIV-1 infection. 4) Immunoblotting and photocatalytic enzyme-linked reaction were used to study the effect of myricetin on the formation of SEVI. The interaction site between myricetin and SEVI was predicted by computer-simulated molecular docking method. 2. The effect of myricetin on SEVI-mediated HIV-1 infection enhancement 1) The effect of myricetin on SEVI-mediated virus infection enhancement was observed by flow cytometry and immunofluorescence microscopy. 2) Virus and Zeta-potential potential potential were used to study the effect of myricetin on SEVI. Effects of myricetin on the formation of amyloid fibers in semen and the enhancement of viral infection 1) Effects of myricetin on amyloid fibers formation in semen were detected by Thioflavin T fluorescence assay. 2) Effects of myricetin on the enhancement of viral infection in semen were detected by TZM-b1 cell infection model. 3) Detecting the synergistic anti-HIV-1 effect of myricetin and various ARVs drugs in semen environment. 4. Degradation of mature SEVI amyloid fibers by myricetin 1) Degradation of mature SEVI amyloid fibers by Thioflavin T fluorescence, Congo red staining and transmission electron microscopy. 2) Detection of myricetin on mature SEVI amyloid fibers by circular dichroism The effect of myricetin on the secondary structure of SEVI amyloid fibers was investigated by TZM-b1 cell infection model. The results showed that myricetin could inhibit the formation of SEVI amyloid fibers in a dose-dependent manner and 75 ug/ml myricetin could completely inhibit the formation of SEVI amyloid fibers in 48 h. The results of circular dichroism showed that myricetin could significantly inhibit the formation of typical secondary structure beta-sheet lamellae of amyloid fibers. The inhibition mechanism of myricetin was found to inhibit the formation of early SEVI oligomers and the whole process of fiber elongation. Myricetin can inhibit SEVI-mediated HIV-1 infection by reducing the positive potential of SEVI and inhibiting the adsorption of SEVI to HIV-1 particles. 3. Myricetin can inhibit the enhancement of SEVI-mediated HIV-1 infection in a dose-dependent manner. In the presence of semen, myricetin can produce synergistic anti-HIV-1 activity with a variety of antiretroviral drugs, such as zidovudine (AZT), raltegravir (RAL), maraviroc (MAR), tenofovir (TNF), nevirapine (NVP), efavirenz (EFV), etc. The CI index was less than 0.5.4. Myricetin could rapidly degrade the mature SEVI amyloid fibers, thus blocking the SEVI-mediated enhancement of HIV-1 infection. Conclusion: Myricetin could significantly inhibit the whole process of oligomer formation and fiber elongation in early SEVI, and inhibit the adsorption of SEVI on HIV-1 virus particles. In addition, myricetin can inhibit the enhancement of semen-mediated HIV-1 infection and synergistic anti-HIV-1 effect with a variety of ARVs drugs in the presence of semen. Myricetin can also degrade mature SEVI amyloid fibers, thereby destroying SEVI-mediated HIV-1 infection enhancement. Myricetin has anti-HIV-1 activity, so it is very likely to be developed as a bifunctional candidate microbicide with high anti-HIV-1 activity and anti-SEVI-mediated HIV-1 infection enhancement.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R96
【参考文献】
相关期刊论文 前2条
1 Joseph R Roberts;Lacey L Siekas;Andrew M Kaz;;Anal intraepithelial neoplasia: A review of diagnosis and management[J];World Journal of Gastrointestinal Oncology;2017年02期
2 段江曼;裘佳寅;谭穗懿;刘叔文;李琳;;精液源性病毒感染增强因子SEVI-HIV性传播中的重要因素[J];病毒学报;2012年01期
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