| 题名 | 天花粉蛋白抗HIV-1构效关系研究及机制探讨 |
| 作者 | 王建华 |
| 学位类别 | 博士 |
| 答辩日期 | 2005-06 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 郑永唐 |
| 关键词 | 核糖体失活蛋白 天花粉蛋白 人免疫缺陷病毒 抗病毒活性 构效关系 |
| 其他题名 | The Structure - Functional Relationship and the Mechanism of Trichosanthin on HIV |
| 中文摘要 | 天花粉蛋白（Trichosanthin，TCS）是从葫芦科植物括楼（TrichosanthesKiriloii）球根中提取的一种由247个氨基酸组成的I型核糖体失活蛋白（RibosomeInactivatingProtein，RIP）。TCS具有广谱的生物学和药学活性，包括抗肿瘤、免疫抑制、中期引产以及抗病毒活性，TCS还具有抗白血病和淋巴瘤的作用。TCS能够抑制HI卜1在急性感染的T淋巴细胞和慢性感染的巨噬细胞中的复制。TCS抗HIV的机制还不清楚，一般认为与R1（RibosolneInactivating）活性有关。TCS的毒副作用限制了它在抗HIV／AIDS临床上的进一步应用。人们期望通过改造或修饰，在保留TCS抗HIV活性的同时，降低其神经毒副作用及所引起的变态反应。因此，研究TCS的抗HIV-1作用机制及构效关系，有利于拓宽TCS的临床应用适应症，也对RIP类化合物基础研究和应用研究具有重要的指导作用。本论文在实验室己有研究工作的基础上，对TCS抗HIV-1作用、机制及构效关系进一步研究。首先，采用MTT比色法检侧TcS对人T淋巴细胞系C8166、HIV-1慢性感染细胞系H9／HIV-1IIIB细胞毒性作用以及采用台盼蓝染色法检测了TCs对刺激转化的人外周血单个核细胞（PeriphejralBloodMononuclearCen，PBMC）的细胞毒性作用；以合胞体形成抑制实验检测了TCS对实验株HIV-1ms诱导C8166细胞致细胞病变的抑制作用；以捕捉HIV-1p24抗原ELISA方法检测TCS对实验．株HIV-1IIIB在急性感染C8166，细胞和慢性感染Hg细胞中复制的抑制作用、临床分离株HIV-1KMO18在PBMC中复制的抑制作用、耐药株HIV-174v在C8166细胞中复制的抑制作用。其次，利用荧光实时定量RT-PCR检测了TCS对HIV-IIIB吸附和融合宿主细胞的抑制作用；采用高效液相色谱（HighPerformanceLiquidChromatograPhy，HPLC）检测了TCS脱HIV-IRNA腺嘿呤作用；另外还检测了TCS对病毒颗粒的直接杀伤作用、TCS对HIV感染和未感染细胞融合的抑制以及对HIV重组逆转录酶活性的抑制作用。最后，利用以蛋白工程技术构建的14个TCD突变体研究其抗HIV的构效关系，其中活性中心突变体：结果表明，TCS不仅能够有效抑制实验株HIV-1mB在C8166细胞中的复制和HIV-1ms诱导宿主细胞的病变作用，还能抑制临床分离株HIV-1KM018在PMBC中的复制和耐药株HIV-174v在C8166细胞中的复制，但TCS对HIV-1在慢性感染H9细胞中的复制无直接抑制作用。TCS不能抑制HIV-1进入宿主细胞；对感染细胞和未感染细胞的融合没有抑制作用；TCS也不能抑制HIV-1重组逆转录酶活性；TCS对病毒颗粒的直接杀伤作用不大；但TCS能够使裸露的HIV-1RNA脱腺漂呤，可能TCS的脱缥岭活性或其它酶活性直接损伤病毒或者病毒感染细胞的核酸，而胞质腺嗦岭含量的增高则可以导致线粒体膜电位的降低、细胞色素C的释放、活性氧的增加和抗凋亡因子表达的下降，结果使感染细胞更多的凋亡，这可能是TCS抗HIV的一个机制。结果也表明，活性中心突变体TCSM（120-123）与TCSE160A/E189A，在失去绝大部分R工活"性的同时，也几乎完全失去抗HIV活性。、而另一个活性中心突变体TCSR122G，RI活性下降1的倍，却仍保留一定的抗HIV活性。TcSC末端删除突变体（TCSC2，TCSC4和TCSC14）抗HIV活性的下降（1.4-4.8倍）与其R1活性呈平行下降（1.2-3.3倍）。这些结果表明TCS抗HIV-1活性与其R1活性显著相关，但似乎又不是唯一的决定因素，因为我们发现二个分别在C末端加上末端19个氨基酸延伸肤或KDEL信号肤的突变体TCS饥触与TCSKDEL，虽然保留全部的RI活性，但却几乎完全失去抗HIV活性，表明有其它机制介入了TCS的抗HIV-1活性。TCS抗原决定簇位点突变后对TCS抗HIV-1活性没有显著影响，但当在抗原决定簇突变体所引入的Cys残基上加上PEG漱后，这些突变体则显著降低了抗HIV-1的活性。 |
| 英文摘要 | Trichosanthin (TCS) is a type I ribosome inactivating protein (REP) isolated from the root tubers of the Chinese medicinal herb Trickosanthes kirilowii Maxim. TCS possesses a wide spectrum of biological and pharmacological properties, including anti-tumor, immunosuppressive, abortifacient and antiviral activity. TCS have been found preferentially inhibited replication of human immunodeficiency virus type 1 (HIV-1) in both acutely infected T-lymphoblastoid cells and chronically infected macrophages in vitro. The mechanism is still unknown, but generally believed that the antiviral activity of TCS is related to the ribosome inactivating (RI) activity. The side effects of TCS have hindered its further clinical trial as anti-HIV/AIDS reagent. It is necessary to modify TCS molecule in order to decrease its neurotoxicity and anaphylactic side effects. Therefore the study of structure-functional relationship of TCS anti-HIV-1 will be helpful and favorable for the foundational and applied investigations of RIPs, and will widen the clinical trial of TCS. The anti-HIV-1 activity, the potential mechanism and the structure-functional relationship were studied in this dissertation. Firstly, cytotoxicity of TCS on C8166 T lymphocyte cell line, HIV-1 chronically infected H9 cell line (H9/HIV-lmB), and peripheral blood mononuclear cell (PBMC) were tested by using MTT colorimetric assay or trypan blue exclusive assay; The inhibition of TCS on HIV-1 induced cytopathic effect was quantitated by the syncytium formation; and the replication inhibition of TCS on ffiV-lmB in C8166, drug-resisted strain HIV-I74V in C8166 and clinically primary isolated strain HIV-1 KMOH in PBMC were assayed by using capture p24 antigen ELISA. Secondly, the inhibition effect of TCS on HIV attachment and fusion steps were tested by using fluoresence-based real-time quantify RT-PCR; The depurination effect of TCS on naked HIV-1 RNA was assayed by using HPLC system; What is more, the direct killing effect of TCS on HIV-1 particle, the inhibition on HIV-1 recombination reverse transcriptase (RT) activity and the inhibition on fusion of HIV infected cells with uninfected cells were tested. Finally, the stracture-fimctional relationship of TCS anti-HTV-l was investigaed by using 14 TCS mutants, including TCS acitive site mutants (TCSM(12W23> TCSEI C-tenninus mutants ( TCSQ, TC3C*" TCSCM, TCSKDEL , TCScww), and antigen determinant site mutants (Q219C , K173C " S7C) and their PEGylation forms(PEG20K-Q219C, PEG2GK-K173C, PEGIOK-STC). The results showed that TCS efficiently inhibited laboratory strain HIV-1ms replication in C8166 cells and the cytopathic effect of host cells, also TCS displayed inhibition effect in HTV-IKMOI S replication in PBMC and HIV-174V replication in C8166 cells. Though TCS exhibited a direct killing effect on HIV-1 chronically infected H9 ceEs, there was no direct inhibition on HIV-1 replication in such cells. Another, all of the inhibition effects of TGS on entry of HIV into host cells, the fusion of infected and uninfected cells, and rRT activity, were not observed. What is more, TCS could not directly kill HIV-1 particle. TCS could depurinate naked HIV-1 RNA. Maybe TCS could recognize other structures located in HIV-1 RNA besides of the classical stem-loop-like structure which always was recognized for the depurination of 28S rRNA. The enrichment of depurinated adenine by TCS in cytoplasm of infected cells would contribute to the drop of of mitochondria A^Pm, the release of cytochrome C, the addition of reactive oxygen species and the decrease of anti-apoptosis factors, which would lead to apoptosis. The results also showed that the active site mutants TCSM < 120-123 > and TCSEI6OA/EI89 with greatest decrease in RI activity, almost lost all of the anti-HIV-1 activity, another active site mutant TCSR1220, which exhibited a 160-fold drop in RI activity, retained some anti-HIV-1 activity. The RI activity of the C-terminal deletion mutants (TCSQ, TCS04 and TCSCM) decreased by 1.2-3.3 fold with parallel downshifting of its anti-HIV-1 activity (1.4-4.8 fold). These results from these studies suggested that RI activity of TCS might have significant contribution to its anti-HIV-1 property. While the anti-HIV-1 activity of TCS was not entirely dependent on the RI activity, because there were two TCSCi9aa and TCSKDEL, having 19 amino acids extension and a KDEL signal sequence; added.to the C-terminus, retained all RI activity but subsequently lost most of the anti-HIV-1 activity. Maybe there were another mechanisms involved in TCS anti-HIV-1 action. The mutant of TCS antigen determinant sites had no significant effect on its anti-HIV-1 activity, while the site-directed PEGylation of TCS had led to a substantial drop in its anti-HIV-1 activity. |
| 语种 | 中文 |
| 公开日期 | 2010-10-15 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6171]  |
| 专题 | 昆明动物研究所_分子免疫药理学 |
| 推荐引用方式 GB/T 7714 | 王建华. 天花粉蛋白抗HIV-1构效关系研究及机制探讨[D]. 北京. 中国科学院研究生院. 2005. |
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