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

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105～106 v.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1.0×1011 v.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.

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

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 10SUP>5/SUP>-10SUP>6/SUP>/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 1.0×10SUP>11/SUP> v.g./ml. After infected with rAAV EM>in vitro/EM>, U2OS cells growth was inhibited significantly. 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 GSUB>1/SUB> arrest were observed. Conclusion The recombinant adeno associated virus