反义细胞周期蛋白B1重组AAV抑制骨肉瘤U2OS细胞增殖和诱导凋亡  

Recombinant adeno-associated virus containing antisense nucleotide against cyclin b1 inhibits osteosarcoma cell U2OS growth and induces apoptosis

作　　者：卓建新 李翔[2] 项大业 陈日曙 张伶[3] 

机构地区：[1]浙江省乐清市第二人民医院,浙江温州325608 [2]浙江省人民医院,杭州310014 [3]中国人民解放军成都军区总医院肿瘤科,成都610083

出　　处：《解剖学报》2009年第3期414-418,共5页Acta Anatomica Sinica

基　　金：浙江省医药卫生科学基金资助项目(2007A180);浙江省自然科学基金资助项目(y207353)

摘　　要：目的研究携带反义细胞周期蛋白B1（CCNB1）的腺相关病毒载体（rAAV-AS-CCNB1/Neo）对骨肉瘤细胞U2OS的细胞增殖抑制和诱导凋亡的影响。方法将构建的重组AAV载体pd16-95-AS-CCNB1/neo和AAV/腺病毒辅助质粒pSH3共转染HEK293细胞,得到表达AS-CCNB1的重组AAV,纯化并浓缩病毒,测定病毒滴度。用重组AAV感染人骨肉瘤U2OS细胞（感染复数为10^5-10^6v.g./细胞）,采用Western blotting、RT-PCR、四甲基偶氮唑盐（MTT）法、流式细胞术等检测或观察重组AAV对细胞周期蛋白B1表达,瘤细胞体外增殖、凋亡和细胞周期的影响。结果成功构建rAAV-AS-CCNB1,病毒滴度为1.0×10^11v.g./ml,体外感染U2OS细胞后可明显降低其增殖活性,Western blotting和RT-PCR提示,rAAV-AS-CCNB1可抑制细胞周期蛋白B1表达,流式细胞术提示,细胞出现明显凋亡和G1期阻滞。结论反义CCNB1重组AAV具有明显的抗骨肉瘤U2OS细胞体外增殖作用,其作用可能与其诱导细胞凋亡和周期阻滞有关。Objective To investigate the effect of recombinant adeno-associated virus （rAAV） containing antisense nucleotide against cyclin B1 （AS-CCNB1） on osteosarcoma cells in vitro. Methods The rAAV was produced by Co-transfecting HEK293 cell line with the rAAV plasmid pd16-95-AS-CCNB1/neo and AAV/Adenoviral helper plasmid pSH3. The rAAV particles expressing AS-CCNB1 were purified and concentrated. Subsequently the virus titer was assayed by dot-bloting analysis. Then, human osteosarcoma U2OS cells were infected by rAAV at the pfu of 10^5-10^6/cell titer. CCNB1 expression was detected by Western blotting and RT-PCR methods, human osteosarcoma U2OS cells growth curve was assayed by MTT, cell cycle and apoptosis were detected by FACS. Results The rAAV-AS-CCNB1 plasmid was successfully constructed and the virus titer is up to 1.0 × 10^11 v.g./ml. After infected with rAAV in vitro, U2OS cells growth was inhibited significandy. Both Western blotting and RT-PCR assay showed that CCNB1 expression of U2OS cells was down-regulated by rAAV-AS-CCNB1, and cell apoptosis and G1 arrest were observed. Conclusion The recombinant adeno-associated virus （rAAV） containing antisense nucleotide against cyclin B1 （AS-CCNB1） showed the ability of inhibiting of human osteosarcoma U2OS cells growth, inducting of apoptosis and cell cycle arrest.

关 键 词：反义CDNA 细胞周期蛋白B1 骨肉瘤 腺相关病毒 流式细胞术 人 

分 类 号：R73[医药卫生—肿瘤]