楊梅素拮抗SEVI介導(dǎo)的HIV-1感染增強(qiáng)作用及其機(jī)制研究
發(fā)布時間：2018-09-06 16:17
【摘要】：研究目的:殺微生物劑(Microbicides)是一類含有抗HIV成分的凝膠、乳脂、栓劑、藥膜或海綿,一般在性交前置入使用者的陰道或肛門內(nèi),用于預(yù)防HIV和其它性傳播病原體的傳播。由于其是一種可被女性控制的有效避免HIV感染的手段,因此成為預(yù)防艾滋病的一種新選擇。但遺憾的是,迄今為止尚無殺微生物劑類藥物問世。讓人費(fèi)解的是,多聚陰離子類殺微生物劑在臨床前動物實(shí)驗(yàn)中均顯示較好的預(yù)防HIV效果,但在臨床試驗(yàn)階段中卻屢屢失敗。導(dǎo)致殺微生物劑臨床試驗(yàn)失敗的原因是什么呢?由于動物攻毒過程中一般都直接采用PBS稀釋病毒,而殺微生物劑用藥部位多為陰道或直腸,不能忽視精液對殺微生物劑藥效的影響。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,可以形成淀粉樣纖維,并顯著增強(qiáng)HIV感染,被命名為精液源性病毒感染增強(qiáng)因子(semen-derived enhancer of viral infection,SEVI)。隨后,來源于精囊凝固蛋白的多肽片段SEM1和SEM2,以及同樣來自酸性磷酸酶蛋白的N末端多肽片段PAP85-120先后被報道。上述這些精液源性的多肽片段均能形成淀粉樣纖維,并增強(qiáng)HIV感染,極有可能是導(dǎo)致多聚陰離子類殺微生物劑臨床試驗(yàn)失敗的主要原因。據(jù)此,精液源性的淀粉樣纖維成為抗HIV感染藥物研發(fā)的新靶點(diǎn)。如果利用藥物阻止精液源性淀粉樣纖維的形成或阻斷HIV與纖維結(jié)合,理論上可降低淀粉樣纖維對病毒的感染增強(qiáng)作用,從而降低HIV的感染率。其中,SEVI為最早發(fā)現(xiàn)的精液源性淀粉樣纖維,其結(jié)構(gòu)穩(wěn)定,在精液中含量較多,研究也最為深入,因此本文擬將SEVI作為主要的藥物作用靶點(diǎn)進(jìn)行研究。由于殺微生物劑必須具備高效、安全、產(chǎn)業(yè)化生產(chǎn)以及成本低等特性,而我國中草藥天然資源眾多,價格低廉,主要用于藥品、保健食品、煙草、化妝品的原料或輔料等。其中,楊梅科楊梅屬植物楊梅的黃酮類有效成分楊梅素(Myricetin,Myr)被報道對Aβ,Tau蛋白等多種淀粉樣纖維的形成具有抑制作用。因此,我們推測楊梅素對精液源性淀粉樣纖維的形成也具有同樣的抑制作用。本項(xiàng)目以SEVI為藥物作用靶點(diǎn),通過一系列生物物理及生物化學(xué)手段評估楊梅素對SEVI淀粉樣纖維形成及其功能的影響,深入探討楊梅素拮抗SEVI介導(dǎo)的HIV-1感染增強(qiáng)作用及作用機(jī)制。同時,楊梅素本身具有較強(qiáng)的抗HIV-1病毒活性。因此,楊梅素極有可能被開發(fā)成為一種既具有高效抗HIV-1活性又可拮抗SEVI介導(dǎo)的HIV-1感染增強(qiáng)作用的雙功能候選殺微生物劑,具有較大的臨床應(yīng)用價值。研究方法:1.楊梅素對SEVI淀粉樣纖維形成的影響及其作用機(jī)制1)采用硫黃素T熒光法、剛果紅染色法及透射電鏡法檢測楊梅素對SEVI淀粉樣纖維形成的作用。2)采用圓二色譜法檢測楊梅素對SEVI二級結(jié)構(gòu)β-sheet形成的影響。3)選用HIV-1感染克隆株及TZM-b1細(xì)胞感染模型,檢測經(jīng)楊梅素處理后形成的SEVI對HIV-1病毒的感染增強(qiáng)作用。4)采用免疫印跡法和光催化酶聯(lián)反應(yīng),研究楊梅素對SEVI形成階段的影響。5)采用計算機(jī)模擬分子對接法,預(yù)測楊梅素與SEVI的相互作用位點(diǎn)。2.楊梅素對SEVI介導(dǎo)HIV-1感染增強(qiáng)作用的影響1)采用流式細(xì)胞術(shù),免疫熒光顯微鏡觀察楊梅素對SEVI介導(dǎo)病毒感染增強(qiáng)作用的影響。2)Virus 及Zeta-potential電勢的方法研究楊梅素對SEVI與HIV-1病毒顆粒吸附作用的影響。3.楊梅素對精液中淀粉樣纖維的形成及病毒感染增強(qiáng)作用的影響1)采用硫黃素T熒光法檢測楊梅素對精液中淀粉樣纖維形成的影響。2)采用TZM-b1細(xì)胞感染模型檢測楊梅素對精液增強(qiáng)病毒感染能力的影響。3)檢測精液環(huán)境中楊梅素與各類ARVs藥物協(xié)同抗HIV-1作用。4.楊梅素對已形成的成熟SEVI淀粉樣纖維的降解作用1)采用硫黃素T熒光法、剛果紅染色及透射電鏡法檢測楊梅素對成熟的SEVI淀粉樣纖維的降解作用。2)采用圓二色譜法檢測楊梅素對成熟的SEVI淀粉樣纖維二級結(jié)構(gòu)β-sheet的影響。3)采用TZM-b1細(xì)胞感染模型檢測楊梅素對降解后SEVI增強(qiáng)病毒感染能力的影響。研究結(jié)果:1.楊梅素能劑量依賴性地抑制SEVI淀粉樣纖維的形成,75 μg/ml的楊梅素在48 h內(nèi)能完全抑制淀粉樣纖維的形成。圓二色譜結(jié)果表明,楊梅素能顯著抑制淀粉樣纖維的典型二級結(jié)構(gòu)β-sheet片層的形成。對其抑制作用環(huán)節(jié)的研究發(fā)現(xiàn),楊梅素可抑制SEVI早期寡聚物形成和纖維延長的全過程。計算機(jī)模擬分子對接發(fā)現(xiàn),楊梅素可通過共價結(jié)合作用,與PAP248-286多肽的Leu-258、Gln-259、Val-264、Leu-268、Met-271 及 Arg-273 等位點(diǎn)特異性結(jié)合。2.楊梅素通過降低SEVI表面正電勢,抑制SEVI對HIV-1病毒顆粒的吸附作用,從而拮抗SEVI介導(dǎo)的HIV-1感染增強(qiáng)作用。3.楊梅素能劑量依賴性地抑制精液中淀粉樣纖維的形成,5 μg/ml的楊梅素可特異性阻斷精液所介導(dǎo)的HIV-1感染增強(qiáng)作用。在精液存在下,楊梅素能與多種抗逆轉(zhuǎn)錄病毒藥物,如 zidovudine(AZT)、raltegravir(RAL),maraviroc(MAR),tenofovir(TNF),nevirapine(NVP),efavirenz(EFV)等,產(chǎn)生協(xié)同抗 HIV-1 活性,其拮抗系數(shù)CI指數(shù)均小于0.5。4.楊梅素可迅速降解已經(jīng)形成的成熟SEVI淀粉樣纖維,從而阻斷SEVI介導(dǎo)的HIV-1病毒感染增強(qiáng)作用。結(jié)論:楊梅素能顯著抑制SEVI早期寡聚物形成和纖維延長的全過程,抑制SEVI對HIV-1病毒顆粒的吸附作用拮抗SEVI介導(dǎo)的HIV-1感染增強(qiáng)作用。此外,楊梅素可抑制精液介導(dǎo)的HIV-1感染增強(qiáng)作用,并能在精液存在下與多種ARVs藥物發(fā)生協(xié)同抗HIV-1作用。同時,楊梅素也可以降解成熟的SEVI淀粉樣纖維,從而破壞SEVI介導(dǎo)的HIV-1病毒感染增強(qiáng)作用。由于楊梅素本身也具有抗HIV-1病毒活性,因此,楊梅素極有可能被開發(fā)成為一種既具有高效抗HIV-1活性又可拮抗SEVI介導(dǎo)的HIV-1感染增強(qiáng)作用的雙功能候選殺微生物劑,具有較大的臨床應(yīng)用價值。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R96
本文編號：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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R96
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
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;譚穗懿;劉叔文;李琳;;精液源性病毒感染增強(qiáng)因子SEVI-HIV性傳播中的重要因素[J];病毒學(xué)報;2012年01期
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