Suramin作为预防艾滋病多效杀微生物剂的研究

发布时间：2018-03-03 20:39

本文选题：Suramin　切入点：淀粉样纤维　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景:艾滋病(Acquired immunodeficiency sydrom,AIDS)是由人类免疫缺陷病毒(Human immunodeficiency virus type 1,HIV-1)引起的一种免疫能力丧失的传染性疾病,严重威胁着人类的生命和健康。全球范围内,新感染病例数居高不下,其中80%新感染病例来自性传播,性传播已成为HIV-1最主要的传播途径。杀微生物剂是种有效预防HIV-1性传播的重要手段。但迄今为止,仍未开发出有效的杀微生物剂。精液来源的病毒增强因子(SEVI)能削弱杀微生物剂的抗病毒活性同时显著促进病毒的感染。SEVI是前列腺酸性磷酸酶(PAP)裂解的多肽片段PAP248-286自我组装形成的淀粉样纤维。SEVI的阳离子特性能消除病毒和细胞的静电排斥,增大病毒和靶细胞接触几率,从而增强HIV-1感染。因此,SEVI是杀微生物剂的一个重要靶点。我们前期研究表明,HIV-1进入抑制剂ADS-J1既能拮抗SEVI,与抗病毒药物合用具有协同抗病毒效果。但由于ADS-J1具有潜在的致癌性,限制了它的临床应用。我们在ADS-J1类似物中筛选到一个本身具有抗HIV-1活性的化合物Suramin,它是临床治疗锥虫病的经典老药,具有强大的药理活性:抑制蛋白酶和阻断生长因子等。前期研究表明,Suramin能抑制多肽PAP248-286形成SEVI,本文将深入研究Suramin抑制及拮抗SEVI的作用及机制;由于Suramin本身具有抗HIV-1活性,我们也将探讨其与抗病毒药物合用协同抗病毒效果,期望将Suramin开发为杀微生物剂或其辅助用药,更好地预防HIV-1性传播。研究目的从具备拮抗HIV-1性传播过程作用多靶点能力的角度阐述Suramin用于预防HIV-1性传播的新的可能及机制,从老药新用的角度寻求扩大Suramin临床适应症,为预防HIV-1性传播的杀微生物剂研发提供新的研究思路。研究方法1.刚果红染色、透射电镜、激光共聚焦显微镜、圆二色谱、Western blot和病毒感染实验方法检测Suramin对SEVI形成及纤维本身的影响。2.病毒感染实验检测Suramin合用抗逆转录病毒药物(ARVs)的抗病毒效果。3.XTT法检测Suramin对生殖道和淋巴细胞的毒性作用。通过家兔阴道粘膜刺激性实验,Suramin凝胶剂进行安全性初步评价,其中包括组织病理学检查、ELISA法检测阴道灌洗液细胞因子表达水平和PCNA免疫组化评价家兔阴道组织的炎症。4.多肽PAP248-286与Suramin进行分子动力学(MD)模拟,从分子动力学角度证明Suramin抑制SEVI形成机制。研究结果1.Suramin能浓度依赖性抑制多肽PAP248-286纤维化;Suramin能结合SEVI,阻断它和HIV-1的结合,降低SEVI促进HIV-1的感染能力。2.Suramin在精液中联合ARVs可以抑制CCR5受体和CXCR4受体嗜性HIV-1,50%抑制率时的联合指数CI值范围为0.233-0.739。3.Suramin在体外对生殖道细胞和淋巴细胞基本没有毒性作用,它对各细胞系的CC50均大于1000 μM。组织病理学检查结果显示Suramin凝胶组的阴道粘膜上皮细胞均较完整,固有层组织仅轻度水肿;Suramin凝胶不会促进炎症因子的释放;Suramin凝胶组的阴道上皮细胞和基质细胞PCNA阳性表达率与生理盐水组无显著性差异。4.MD模拟表明分子结构非常相似的3个化合物与PAP248-286结合模式不同,分子结构趋向于立体的Suramin能把PAP248-286完全包裹住,另外其改变PAP248-286单体结构的程度最大:Suramin几乎与PAP248-286 上所有的氨基酸有氢键或疏水作用:氢键有19个,疏水作用数为45。研究结论1.Suramin能紧密结合多肽PAP248-286,抑制其自我聚合形成淀粉样纤维SEVI。2.Suramin阻断SEVI和HIV-1结合,拮抗其增强病毒感染能力。3.Suramin在精液中分别联合不同作用机制的ARVs均具有协同抗病毒作用,且对生殖道细胞和淋巴细胞基本无毒性作用,对家兔阴道无明显致炎作用,有较高的安全性。
[Abstract]:Background: AIDS (Acquired immunodeficiency, Sydrom, AIDS) is by the human immunodeficiency virus (Human immunodeficiency virus type 1, HIV-1) caused a loss of immunity to infectious diseases, serious threat to human life and health. Globally, the number of new infections is high, of which 80% new infections from spreading. Sexual transmission has become the most important route of transmission of HIV-1. A microbicide is an important means for effective prevention of sexual transmission of HIV-1. But so far, has not yet developed effective microbial kill agent. Semen derived virus enhancement factor (SEVI) can weaken the microbicide antiviral activity also significantly promote.SEVI virus infection is prostatic acid phosphatase (PAP) characteristics of cationic polypeptide fragment PAP248-286 cleavage of the self-assembly of amyloid fibrils formed by the.SEVI can eliminate the virus and cell The electrostatic repulsion, the risk of viral and target cell contact increases, thereby enhancing the infection of HIV-1. Therefore, SEVI is an important target for microbicides. Our previous study showed that HIV-1 can antagonize SEVI ADS-J1 entry inhibitors, in combination with antiviral drugs has synergistic antiviral effect. But because ADS-J1 has potential carcinogenicity, limiting the clinical the application of it. We screened ADS-J1 analogues in a compound with anti HIV-1 activity of Suramin, it is a classic old drug treatment of trypanosomiasis, has strong pharmacological activity: inhibition of protease and blocking growth factor. Previous studies have shown that Suramin can inhibit the formation of peptide PAP248-286 SEVI, effect and mechanism this paper will deeply Research on Suramin inhibition and antagonism of SEVI; because Suramin itself has anti HIV-1 activity, we will also discuss the Chinese medicine combined with synergistic antiviral effect, Expect to exploit Suramin as a microbicide or auxiliary medicine for better prevention of sexual transmission of HIV-1. The purpose of the study on the Suramin from antagonism to HIV-1 communication process multi-target capability perspective for possible new mechanism and prevention of sexual transmission of HIV-1, seeking to expand Suramin clinical indications from the angle of the new old drugs and provide a new idea for the microbicide to prevent sexual transmission of HIV-1. Methods 1. Congo red staining, transmission electron microscopy, laser confocal microscope, round two chromatography, detection of Suramin on the formation of SEVI and blot test method of Western virus infection and the effect of fiber.2. virus infection assay Suramin combined antiretroviral drugs the detection of Suramin (ARVs) toxicity of the antiviral effect of.3.XTT method on genital and lymphocytes. The rabbit vaginal mucosa irritation test, Suramin gel into. Preliminary evaluation, including histopathological examination, detection of vaginal lavage cytokine ELISA expression and immunohistochemistry of PCNA.4. peptide PAP248-286 and Suramin in the assessment of inflammatory rabbit vaginal tissue by molecular dynamics (MD) simulations show that Suramin inhibits SEVI formation mechanism from the view of molecular dynamics. The research results of 1.Suramin concentration dependence inhibition of polypeptide PAP248-286 fibrosis; Suramin can bind to SEVI, blocking its combination with HIV-1, reduce HIV-1 infection combined with ARVs promote the ability of.2.Suramin in semen SEVI index can be combined inhibition of CCR5 receptor and CXCR4 receptor tropism HIV-1,50% inhibition rate when the CI value is in the range of 0.233-0.739.3.Suramin in vitro on reproductive tract cells and lymphocytes basically no toxic effects. It is on the cell line CC50 was greater than 1000 M. histopathological examination showed vaginal gel group Suramin Epithelial cells were relatively complete propria tissue only mild edema; Suramin gel does not promote the release of inflammatory factors; Suramin gel group PCNA vaginal epithelial cells and stromal cells and the positive expression rate of saline group had no significant difference between the.4.MD simulation shows that the molecular structure is similar to 3 compounds and PAP248-286 binding mode, molecular the three-dimensional structure tends to Suramin the PAP248-286 can completely wrap, also the change of PAP248-286 monomer structure maximum: Suramin and PAP248-286 almost all amino acids have hydrogen bonds and hydrophobic interaction of hydrogen bonds 19, hydrophobic interaction number 45. conclusions 1.Suramin can combine polypeptide PAP248-286, inhibiting its self polymerization to form starch kind of fiber SEVI.2.Suramin blocking SEVI and HIV-1 combination, enhance the ability of.3.Suramin antagonistic virus infection in semen respectively with different effect The mechanism of ARVs has synergistic antiviral effect, and has no toxic effect on reproductive tract cells and lymphocytes. It has no obvious inflammation effect on rabbit vagina, and has high safety.

【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】：R512.91

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