2型登革病毒诱导RAW264.7细胞凋亡的机制及凋亡对病毒复制的影响  

Dengue virus type 2 induces apoptosis of RAW264.7 cells and effect of apoptosis on virus replication

作　　者：张林[1] 黄俊琪[1] 

机构地区：[1]中山大学中山医学院免疫学研究所教育部热带病防治研究重点实验室,广东广州510080

出　　处：《中国病理生理杂志》2013年第12期2245-2250,共6页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30872350;No.31370870);广东省自然科学基金资助项目(No.S201302001300;No.S2012010009050);广东省科技计划(No.2010B050700008;No.2011B040300022);广州市科技计划(No.2011J4100084);2010年中山大学高校基本科研业务费青年培育项目(No.10ykpy31)

摘　　要：目的:探讨2型登革病毒(DENV2)感染能否诱导RAW264.7细胞凋亡,并初步探讨凋亡对病毒复制的影响。方法:用DENV2感染RAW264.7细胞,MTT检测细胞活性,Hoechst 33342染色检测细胞核变化,Annexin V-FITC/PI双染流式细胞术检测细胞凋亡,Western blotting检测caspase-3和caspase-8活化片段的变化,比色法检测caspase-9活性变化,JC-1染色检测线粒体膜电位变化,Z-VAD-FMK抑制细胞凋亡后以TCID50检测感染细胞上清病毒滴度。结果:DENV2感染RAW264.7细胞24 h、36 h及48 h后细胞活性受到抑制,免疫荧光检测有核固缩现象,流式细胞术检测发现病毒感染诱导了细胞凋亡,Western blotting检测发现活化caspase-3和caspase-8的表达增加,caspase-9活性也增加,JC-1染色发现病毒感染诱导RAW264.7细胞线粒体膜电位降低,用Z-VAD-FMK抑制凋亡后感染细胞上清病毒滴度增加。结论:登革病毒感染可以通过内、外源性途径诱导RAW264.7细胞发生凋亡;凋亡发生抑制了病毒的产生。AIM： To explore the apoptotic pathway in dengue virus type 2 （DENV2）-infected RAW264.7 ceils and to analyze the effect of apoptosis on virus replication. METHODS ： RAW264.7 cells were infected with DENV2. MTF assay was used to detect the cell viability. Apoptosis was assessed by Hoechst 33342 staining and Annexin V-FITC/PI staining. The expression levels of caspase-3 and caspase-8 were determined by Western blotting. The activity of caspase-9 was measured with a colorimetric kit. Mitochondrial membrane potential was evaluated using JC-1 fluorescent staining. TCIDs0 was used to estimate the infectious virion concentration after using Z-VAD-FMK to inhibit apoptosis. RESULTS： The viability of RAW264.7 cells decreased after DENV2 infection at 24 h, 36 h and 48 h. Karyopyknosis in dengue virus- infected ceils was observed. The protein levels of caspase-3 and caspase-8 and the activity of caspase-9 increased in the ap- optotic cells after dengue virus infection. Mitochondrial membrane potential was reduced after dengue virus infection. There was a higher virion concentration in the cell culture medium after inhibition of apoptosis. CONCLUSION： Dengue virus induces apoptosis of RAW264.7 cells. Apoptotic inhibition of RAW264.7 cells facilitates the production of dengue virus. [ KEY WORDS] Dengue virus ; Apoptosis ; RAW264.7 cells

关 键 词：登革病毒 细胞凋亡 RAW264 7 细胞 

分 类 号：R373.33[医药卫生—病原生物学]