新城疫病毒FMW株体外溶瘤作用及其机制分析  

Mechanisms of in vitro anti-tumor effects of a Newcastle disease virus

作　　者：孟松树[1] 白海[1] 胡茂志[1] 刘文博[2] 王莉[2] 吴艳涛[2] 焦新安[1] 范健[1] 

机构地区：[1]扬州大学生物科学与技术学院,扬州225009 [2]扬州大学兽医学院,扬州225009

出　　处：《微生物学报》2009年第4期512-517,共6页Acta Microbiologica Sinica

基　　金：江苏省自然科学基金(BK2006071);江苏省卫生厅项目(H200763)~~

摘　　要：【目的】筛选出能高效抑制多种人肿瘤细胞生长增殖的新城疫(New Castle disease virus,NDV)毒株,为进一步构建重组高效靶向溶瘤毒株奠定基础。【方法】以体外噻唑蓝法测定NDV对A549、SMMC7721等肿瘤细胞及人胚干细胞L-O2、人胚肾细胞HEK293等的生长抑制率,空斑试验确定病毒滴度及感染复数。利用形态学观察、Hoechst荧光染色、流式细胞术及免疫印迹等分析了NDV-FMW诱导肿瘤细胞凋亡的细胞生物学变化及其机制。【结果】从近50株NDV中筛选出NDV-FMW,以20MOI病毒作用A549、SMMC7721等肿瘤细胞48h,细胞生长抑制率达60%,NDV-FMW诱导肿瘤细胞发生凋亡,效应呈时间和剂量的依赖性,凋亡细胞出现核染色质断裂、浓缩及二倍体亚峰,细胞周期阻滞于G0/G1期,此外,病毒感染A549细胞16h后开始检测到活化的Caspase-3裂解片段及PARP裂解大片段。【结论】NDV-FMW株体外能高效抑制肿瘤细胞的增殖,并经Caspase-3途径诱导肿瘤细胞凋亡。FMW株具有自主知识产权,其良好的体外溶瘤能力为进一步探讨体内抗肿瘤及临床试验的进行奠定了基础,并有可能为恶性肿瘤的治疗提供新的生物制剂。[ Objective] To obtain Newcastle disease virus （NDV） strains with high in vitro anti-tumor effect for construction of recombinant NDV for clinical therapy. [ Methods] We used MTT [ 3-（4, 5-dimethyhhiazol-2-yl）-2,5-diphemyhetra-zolium Bromide] assay to examine the anti-tumor effect on A549 and SMMC7721 cells infected by nearly 50 NDV strains. Several assays were used to analyze the apoptosis induced by NDV infection. These assays included：（1） Morphological analysis; （2） Hoechst？ fluorescence dye testing; （3） Flow cytometric analysis, and （4） Western blot. [ Results] We obtained an NDV FMW strain that inhibited cell growth up to 60% at 48 h postinfection with an MOI （ multiplex of infection） of 20. NDV- FMW could elicit apoptosis of infected tumor cells in a dose- and time- dependent manner. We observed the infected A549 and SMMC7721 cells with condensed and fragmented chromatin at 48 h postinfection. Apoptosis peak and hypodiploid cells were revealed by proidum iodide （PI） staining and cell cycle was blocked and arrested in GO/GI phase in tested cells. Furthermore, annexin-V/PI staining showed that the apoptotic rates in SMMC7721 cells were 2.1%, 18.5 %, 23.8 % and 30.4 % after treated with 0, 0.2, 2 and 20 MOI NDV-FMW for 48 h respectively. To elucidate the apoptosis pathways induced by NDV-FMW, we detected the expression of active caspase-3 and cleavages of poly（ADP-ribose） polymerase （PARP）. We demonstrated that caspase-3 in A549 cells was activated early at 16 h postinfection and PARP was cleaved subsequently. [ Conclusion] NDV-FMW had strong in vitro anti-tumor effect on A549 and SMMC7721 cells. Apoptosis of tumor cells induced by NDV-FMW via caspase-3 activation and NDV-FMW could be a potential cancer virotherapy agent.

关 键 词：新城疫病毒 溶瘤病毒 细胞凋亡 

分 类 号：S852.65[农业科学—基础兽医学]