重组溶瘤流感病毒靶向杀伤肝癌细胞的实验研究  

Experiment of recombinant oncolytic influenza virus targecting effects on liver carcinoma cells

作　　者：孙芳[1] 任天宇[1] 严锦[1] 段跃强[2] 王希良[2] 张绍庚[1] 杨鹏辉[1,2] 

机构地区：[1]解放军第三0二医院肝胆外科一中心,北京100039 [2]军事医学科学院微生物流行病研究所,北京100071

出　　处：《传染病信息》2017年第2期86-90,103,共6页Infectious Disease Information

基　　金：国家“863”计划青年科学家专题(SS2015AA020924)

摘　　要：目的拯救获得重组溶瘤流感病毒株rFlu △ NS/HCC,对其全面鉴定并评价特异性杀伤肝癌细胞的效果。方法利用流感病毒反向遗传学(reverse genetics,RG)技术,以A/Puerto Rico/8/34(PR8)为载体,将NS基因部分敲除,构建重组质粒pFlu △ NS1,与流感病毒PR8的7个质粒pHW191-PB2、pHW192-PB1、pHW193-PA、pHW194-HA、pHW195-NP、pHW196-NA、pHW197-M共转染MDCK和COS-Ⅰ细胞,经拯救、筛选、鉴定获得重组溶瘤流感病毒,命名为rFlu △ NS/HCC。重组溶瘤流感病毒株rFlu △ NS/HCC经SPF鸡胚扩大培养、超滤浓缩、蔗糖密度梯度离心纯化等步骤获得重组病毒,通过血凝试验、RT-PCR、电镜观察病毒形态等方法对rFlu △ NS/HCC进行全面鉴定,并通过MTS细胞活力测定、细胞病变检测评价重组溶瘤流感病毒对不同类型肝癌细胞系的杀伤效果。结果成功拯救重组溶瘤流感病毒rFlu △ NS/HCC,血凝效价为2^(9^10),在HpG2细胞上感染病毒滴度log10TCID50/ml为6.5。MTS和结晶紫染色检测结果显示,重组溶瘤流感病毒rFlu △ NS/HCC感染复数(multiplicity of infection,MOI)≥1时,能够显著降低HepG2细胞活力(P均<0.05);MOI≥3时,对SMMC-7721细胞活力影响达显著水平(P均<0.05);而不同MOI值的rFlu △ NS/HCC对正常细胞L02无明显影响。结论重组溶瘤流感病毒rFlu △ NS/HCC能靶向杀伤肝癌细胞,有望为临床肝癌靶向治疗提供新的治疗策略。Objective To investigate the characterization and selective anti-tumor effects of the recombinant oncolytic virus rFlu △ NS/HCC on liver carcinoma cells. Methods By using reverse genetics（RG）, a recombinant oncolytic influenza virus rFlu △ NS/HCC was rescued and identified in the backbone of A/Puerto Rico/8/34（PR8）. rFlu △ NS1 based on partial deletion of NS（deNS1）was constructed, which was cotransfected with the influenza virus remaining seven plasmids of PB2, PB1, PA, HA, NP, NA and M into MDCK and COS-Ⅰcells. Recombinant rFlu △ NS/HCC virus were amplified by SPF chicken embryos, purified by sucrose gradient, then morphological characteristics of virus were detected with electron microscope, hemagglutination titers and viral titers. MTS and cytopathic assay were used to investigate the oncolytic targeting effects of the recombinant rFlu △ NS/HCC virus on liver carcinoma cells and normal liver cells. Results The hemagglutination titers of recombinant rFlu △ NS/HCC virus was 29-10 and the log10 TCID 50/ml was 6.5 of rFlu/RSV/F virus in HepG2 cells. The results were measured by MTS assay and cytopathic assay. The recombinant rFlu △ NS/HCC virus could induce selective cytotoxicity in liver carcinoma cells HepG2 at MOI of 1（P 〈0.01）, and SMMC-7721 at MOI of 3（P 〈0.001）, without effect in the normal liver cell line（L02）. In summary, the recombinant rFlu △ NS/HCC virus with differment MOI didn′t effect the normal liver cell line（L02）. Conclusions The recombinant oncolytic rFlu △ NS/HCC virus can induce selective cytotoxicity in liver carcinoma cells and provide a new theoretical strategy for the clinical therapy of cancer.

关 键 词：流感病毒 溶瘤病毒 肝癌细胞 

分 类 号：R575.2[医药卫生—消化系统]