利托那韦抗HTLV-1病毒侵染及抑制成人T细胞白血病细胞增殖机制研究  

Anti-HTLV-1 Virus Transmission and Inhibition of Cell Proliferation of Adult T-Cell Leukemia by Ritonavir and the Mechanism Study

作　　者：任欣欣 王莹 朱圣宇 张馨逸 王旻然 徐玲玲[1] 赵铁军 REN Xinxin;WANG Ying;ZHU Shengyu;ZHANG Xinyi;WANG Minran;XU Lingling;ZHAO Tiejun(College of Life Science,Zhejiang Normal University,Jinhua 321004,China;School of Medicine,Hangzhou City University,Hangzhou 310015,China)

机构地区：[1]浙江师范大学生命科学学院,浙江金华321004 [2]浙大城市学院医学院,杭州310015

出　　处：《中国药学杂志》2024年第8期676-686,共11页Chinese Pharmaceutical Journal

基　　金：国家自然科学基金项目资助(32370147,31970173);浙江省自然科学基金探索项目资助(LY21C010001)。

摘　　要：目的研究利托那韦(ritonavir)对人类T细胞白血病病毒1型(human T-cell leukemia virus type-1,HTLV-1)病毒侵染及成人T细胞白血病(adult T-cell leukemia,ATL)细胞恶性增殖抑制作用,并探讨其分子机制。方法采用CCK-8和克隆形成实验检测ritonavir对多种ATL细胞增殖的影响;流式细胞术、双荧光素酶报告基因技术、实时定量聚合酶链反应(qPCR)和Western blot检测ritonavir对HTLV-1病毒侵染的影响;流式细胞术检测ritonavir对ATL细胞周期和凋亡的影响。结果Ritonavir能够抑制4种ATL细胞株的增殖及HTLV-1阳性细胞株的克隆性增殖,前者具有明显的量效关系,且对HTLV-1阳性细胞株的抑制作用更明显(P<0.05)。阳性细胞株与JETWT35细胞共培养后立即加入ritonavir,药物可明显下调后者红色荧光蛋白的表达,抑制HTLV-1病毒侵染到受体细胞(P<0.01)。阳性细胞株和Jurkat细胞共培养体系中立即加入ritonavir后,受体细胞中HTLV-1相关病毒基因Tax等mRNA水平均有抑制作用(P<0.01),而12 h病毒侵染完成后再加入ritonavir,则无这些明显影响。Ritonavir剂量依赖性地抑制阳性细胞株ATL-T细胞表面HTLV-1包膜蛋白亚基gp46的表达,从而抑制HTLV-1病毒的产生和侵染(P<0.01)。Ritonavir将细胞阻滞于G1期,并促进细胞凋亡,且HTLV-1阳性细胞株的凋亡率显著高于阴性细胞株(P<0.05或P<0.01)。结论Ritonavir通过降低WT-Luc病毒启动子活力,下调HTLV-1相关病毒基因(Tax、HBZ、Gag、Pol和Env)表达,以及减少阳性细胞表面HTLV-1包膜蛋白亚基gp46表达从而抑制HTLV-1病毒的产生及侵染,并将细胞阻滞于G1期,诱导其凋亡,从多环节有效抑制ATL细胞增殖。OBJECTIVE To observe the inhibitory effect of ritonavir on human T-cell leukemia virus type-1(HTLV-1)transmission and malignant proliferation of adult T-cell leukemia(ATL)cells,and explore its molecular mechanism.METHODS The proliferation and vitality of ritonavir on various leukemic cells were evaluated by CCK-8 and colony formation assay.The effects of ritonavir on HTLV-1 virus transmission were detected by flow cytometry,dual luciferase reporter gene technique,qPCR and Western blot.The effects of ritonavir on the cell cycle and apoptosis of ATL cells were examined through flow cytometry.RESULTS Ritonavir could inhibit the proliferation of four ATL cell lines and the clonal proliferation of HTLV-1 positive cell lines.The former exhibited a significant dose-effect relationship and had a more pronounced inhibitory effect on HTLV-1 positive cell lines(P<0.05).Additionally,the administration of ritonavir immediately after co-culture of HTLV-1 positive cell lines with JETWT35 cells resulted in a significant down-regulation of red fluorescent protein expression in JETWT35 and inhibited the transmission of HTLV-1 virus into recipient cells(P<0.01).Upon immediate addition of ritonavir to the co-culture system of HTLV-1 positive cell lines and Jurkat cells,there was a notable inhibition of HTLV-1-related gene Tax and other genes mRNA in recipient cells(P<0.01);however,no significant effect was observed when ritonavir was added 12 h after virus transmission.Morever,ritonavir demonstrated a does-dependent inhibition of gp46 expression on the cell membrane of the HTLV-1 positive cell line ATL-T,thereby suppressing the production of HTLV-1 virus(P<0.01).Ritonavir impeded cell progression into G1 phase and facilitated apoptosis,with the apoptosis rate of HTLV-1 positive cell lines being significantly greater than that of HTLV-1 negative cell lines(P<0.05 or P<0.01).CONCLUSION Ritonavir exerts inhibitory effects on the production and transmission of HTLV-1 virus by diminishing the activity of WT-Luc virus promoter,suppressing

关 键 词：利托那韦 人类T细胞白血病病毒1型 病毒侵染 增殖 成人T细胞白血病 

分 类 号：R966[医药卫生—药理学]