【作者】 孙利；

【导师】 白雪帆；

【作者基本信息】 第四军医大学 ， 内科学， 2008， 博士

【摘要】 艾滋病(AIDS)从发现至今已有25年,但它在全球所引起的广泛流行,已使4000多万人受到感染,2200多万人失去了生命。目前全球人类免疫缺陷病毒(HIV)感染者总数达到4200万人,如果不扩大采取有效预防措施,在2010年前,世界126个中、低收入国家还将有4500万人感染HIV,其中40%在亚洲和太平洋地区。中国和印度这两个人口最多的国家HIV/AIDS也在蔓延:中国HIV感染者总数已近100万人,如果不能有效控制,今后10年这一数字可能会成倍增加;印度HIV感染者更是达到了397万,而且今后仍有可能大幅增长。目前应用联合高效的抗逆转录病毒疗法(HAART),已在很大程度上降低了HIV-1感染的发病率和死亡率。三类主要的药物——核苷类逆转录酶抑制剂(NRTI)、非核苷类逆转录酶抑制剂(NNRTI)和蛋白酶抑制剂(PI),它们定向作用于逆转录酶和蛋白酶。尽管这些药物可以有效地抑制病毒感染,但由于抗药性HIV-1的出现,以及抗逆转录病毒治疗中出现的许多不良反应,目前仍迫切需要研发新的治疗药物和方法。在各种新的正在研发的抗HIV药物中,趋化因子受体拮抗剂最令人注目,这不仅因为它能够有效地对抗逆转录病毒活性,还能靶向性阻断HIV-1入侵细胞的关键蛋白——CD4+T细胞和巨噬细胞表面的微孔蛋白(即HIV-1辅受体),可以阻断其与病毒包膜糖蛋白(gp120)间的相互作用,从而发挥抗HIV-1感染的治疗作用。因此,关于HIV-1辅受体及其配体——趋化因子RANTES、MIP-1和SDF-1的研究,成为抗HIV-1感染基因治疗基础研究的热点内容。美国Chen SY领导的科研小组研究表明:α类趋化因子SDF-1的细胞内表达可以从表型上剔除CXCR4,从而阻断T嗜性HIV-1的感染;而β类趋化因子RANTES和MIP-1的细胞内表达可以从表型上剔除CCR5,从而阻断M嗜性HIV-1的感染。我科白雪帆教授、张颖博士等在单一配基表达阻断HIV-1研究的基础上,采用细胞内趋化因子(intrakine)技术构建含有HIV-1两类辅受体的配体——趋化因子RANTES和SDF-1的双表达真核载体和逆转录病毒载体;将表达载体转染各类细胞,观察目的基因的表达及表达产物在细胞内与辅受体的结合;病毒感染实验检测辅受体表型剔除对HIV-1膜蛋白诱导合胞体形成、假病毒HIV-CAT的表达及病毒p24抗原活性,观察了淋巴细胞辅受体的表型剔除对HIV-1病毒感染的阻断作用。在基础和应用研究中,作为基因转移的一种新的有效和多用途的工具,慢病毒载体在基因治疗领域展示出可喜的前景。慢病毒载体能转导非分裂细胞,并能维持转基因持久和长期表达。许多细胞类型,如脑、肝、肌肉和造血干细胞,已经成功地转导了携带多种基因的慢病毒载体。同样,慢病毒载体能设计成可表达治疗性抗HIV-1的基因,特异性地靶向于病毒复制的不同阶段。为此,我们应用分子生物学技术构建了含CC-细胞内趋化因子(CC-intrakine,RANTES-K)和CXC-细胞内趋化因子(CXC-intrakine,SDF-K)的慢病毒表达质粒pLenti6/V5-R-K和pLenti6/V5-S-K,并在293FT细胞中建立了慢病毒株,转导CD34+人造血干细胞(hHSC)后,观测了细胞中RANTES和SDF-1蛋白的表达情况及感染HIV-1病毒液后p24抗原分泌水平,以观察CD34+hHSC辅受体的表型剔除对HIV-1病毒感染的阻断作用。实验结果如下:1、酶切和测序表明HIV-1辅受体CCR5、CXCR4的配体RANTES和SDF-1的慢病毒表达质粒pLenti6/V5-R-K和pLenti6/V5-S-K符合其物理图谱,构建是成功的。2、在293FT细胞中分别制备了包含慢病毒表达质粒pLenti6/V5-R-K和pLenti6/V5-S-K的慢病毒株,滴度分别为8.67×105转导单位(TU)/ml和8.56×105转导单位(TU)/ml,此慢病毒株可用于后续研究。3、将慢病毒株分别转染HeLa细胞和免疫磁珠法分离人脐带血得到的CD34+hHSC(流式细胞仪分析纯度为96.8%),间接免疫荧光证实RANTES和SDF-1蛋白可以表达于人宫颈癌HeLa细胞系和CD34+hHSC内。4、慢病毒株转染的CD34+hHSC在感染HIV-1 DP1/27病毒液后第4、7和10d皆可发现显著的p24抗原表达下降(P<0.05),分别减少了51%、58%、60%(包含慢病毒表达质粒pLenti6/V5-R-K的慢病毒株)和50%、57%、58%(包含慢病毒表达质粒pLenti6/V5-S-K的慢病毒株),表明慢病毒表达质粒pLenti6/V5-R-K和pLenti6/V5-S-K转染具有阻断HIV-1病毒复制的作用。综上所述,本文采用细胞内趋化因子技术进行HIV-1两类主要辅受体CCR5和CXCR4的配体——RANTES和SDF-1的慢病毒质粒表达,使两类HIV-1辅受体被阻断,并转染到CD34+人造血干细胞(hHSC)中,试图为将来回输基因修饰的hHSC进而永久抑制HIV-1复制的抗HIV-1基因治疗寻求一个新的突破点。更多还原

【Abstract】 Acquired immunodeficiency syndrome (AIDS) and human immunodeficiency virus (HIV) infection continue to be major global health concerns, with 1,000,000 Chinese citizens infected by the end of 2002. Although highly active antiretroviral therapy (HAART) has led to profound and prolonged reductions in circulating virus levels in many individuals, high cost, side effects and increasingly the emergence of multidrug-resistant virus strains limit the use of HAART. Gene therapy for HIV infection has got rapid progress in recent years. Several strategies have been approved and are being conducted in clinical trial.Human immunodeficiency virus type 1 (HIV-1) entry into target cells is a multi-step process involving the interaction of viral envelope proteins with cell surface receptors, such as the CCR5 and CXCR4 chemokine receptors. CCR5 and CXCR4 are G protein-coupled receptors for CC- and CXC-chemokines, such as MIP-1α, RANTES and SDF-1, and are also the coreceptors of human immunodeficiency virus (HIV). In concert with CD4, CCR5 and CXCR4 mediate the binding of the viral envelope protein gp120 to the cell surface and allow HIV subsequent entry into target cells.The study by Chen SY et al mimicked the natural resistance of the individuals with the genetic CCR5 defect by inactivating CCR5 using a novel intracellular chemokine (intrakine) strategy. The CC-chemokine,RANTES,a ligand for CCR-5, has been targeted to the lumen of endocytoplasmic reticulum (ER) using a KDEL fusion termed RANTES-KDEL and this construct was found to effectively prevent the transport of newly synthesized CCR-5 to the cell surface. The lymphocytes expressing the CC-intrakine (RANTES-K) were resistant to M-tropic HIV-1 infection, while retaining normal cell functions. In an accompanied study, SDF-1a, a ligand for CXCR4, was also targeted to the ER to inactivate CXCR4, and the viable lymphocytes expressing CXC-intrakine (SDF-K) were found to resist T-tropic HIV-1 infection. These results indicate the therapeutic application of this genetic intrakine strategy to control HIV-1 infection.In the previous study by Bai XF and Zhang Y, a novel“intrakine”strategy was utilized to co-inactivate genetically both CCR5 and CXCR4 in HIV-1 target cells. The principle of co-inactivation of CCR5 and CXCR4 was illustrated by targeting the CC-intrakine and CXC-intrakine to the lumen of the endoplasmic reticulum for intracellular blockade of the transport of newly synthesized chemokine coreceptors to the cell surface. They constructed bicistronic eukaryotic vector and retroviral vector, which harbor the genes of HIV-1 coreceptors ligands RANTES and SDF-1. In order to examine co-expression of RANTES and SDF-1 by the bicistronic vector, several cell strains were transfected with various expression vector DNA. Finally, Env-mediated syncytium formation, envelope complementation assay and p24 detection were carried out to detect anti-HIV-1 activity of the co-inactivation of CCR5 and CXCR4. In summary, they utilized an intrakine strategy to co-inactivate both the principal M-tropic and T-tropic HIV-1 coreceptors in target cells. The lymphocytes with the phenotypic knockout of CCR5 and CXCR4 broadly resisted the infection of various HIV-1 viruses. Thus, intrakine provides a promising therapeutic strategy toward the goal of long-term control of HIV-1 infection.Recent years, some new vectors arosed with the prominent merits and of them, lentiviral vectors have emerged as potent and versatile tools of gene transfer for basic and applied research and are able to transduce nondividing cells and maintain sustained long-term expression of transgenes. For this reason, we constructed the HIV-based lentiviral vector expressing RANTES-KDEL and SDF-KDEL, pLenti6/V5-R-K and pLenti6/V5-S-K, and cotransfected them with the ViraPowerTM Packaging Mix (pLP1, pLP2, and pLP/VSVG) into 293FT cells to produce the replication-incompetent lentivirus stocks. Furthermore, we titrated the lentiviral stocks using HeLa cells, and detected the expression of the gene of interest, RANTES and SDF-1, by indirect immumofluorescence. The results are as following:1、The construction of lentiviral expression vectors, pLenti6/V5-R-K andpLenti6/V5-S-K, was confirmed by enzymatic digestion and sequencing. 2、We constructed lentivirus stocks expressing pLenti6/V5-R-K and pLenti6/V5-S-K with the ViraPower? Packaging Mix in a 293 FT cell line and titrated them in HeLa cell line. We measured the titre of the lentivirus stocks, which was 8.67×105 transduced units (TU)/ml and 8.56×105 transduced units (TU)/ml.3、We detected the RANTES and SDF-1 protein in HeLa cells and CD34+hHSC respectively by a Midi-MACS CD34 Isolation Kit(the purity was 96.8% as evaluated by flow cytometry) transfected with pLenti6/V5-R-K- and pLenti6/V5-S-K-expressing lentivirus using goat-anti-human RANTES and SDF-1 antibody with rabbit-anti-goat IgG-FITC secondary antibody. The majority of RANTES and SDF-1 were expressed in the cytoplasm, especially in the perinuclear region.4、The HIV-1 DP1 / 27 strain was amplified in MT4 cells and the virus titer was 10-4.2/ ml (TCID50)/ 10-4.1/ ml (TCID50). After 6d infection, syncytia were observed in the untransfected cells and in the CD34+hHSC transduced with empty plasmid. In contrast, CD34+hHSC transfected with lentivirus expressing pLenti6/V5-R-K and pLenti6/V5-S-K had no syncytia. We also detected p24 antigen levels of cell culture supernatants on the 4th, 7th, and 10th day, with 103 TCID50 HIV-1 DP-infected CD34+hHSC. The cells transfected with pLenti6/V5-R-K and pLenti6/V5-S-K had a significant reduction of HIV-1 DP transmission with a decrease of 51%、50% on the 4th day, 58%、57% on the 7th day and 60%、58% on the 10th day (P<0.05) compared to the untransfected control cells. The cells transfected with empty plasmid had no discernable decrease of p24 antigen (P>0.05) compared to the untransfected control cells.In summary, we utilized an intrakine strategy to inactivate the principal M-tropic and T-tropic HIV-1 coreceptors in target cells. The CD34+hHSC with the phenotypic knockout of CCR5 and CXCR4 broadly resisted the infection of various HIV-1 viruses. Thus, intrakine provides a promising therapeutic strategy toward the goal of long-term control of HIV-1 infection. These findings demonstrated the ability of lentiviral vectors to transduce multiple genes into human hematopoietic progenitor cells, and the potential therapeutic strategies for the treatment of human diseases.更多还原