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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105~106v.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1.0×1011v.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 effects of rAAV-AS-CCNB1 / Neo carrying antisense cyclin B1 (CCNB1) on the proliferation and apoptosis of osteosarcoma U2OS cells. Methods Recombinant AAV vector pd16-95-AS-CCNB1 / neo and AAV / adenovirus helper plasmid pSH3 were co-transfected into HEK293 cells to obtain recombinant AAV expressing AS-CCNB1. The virus was purified and concentrated to determine the virus titer. Human osteosarcoma U2OS cells (105-106 v.g. / cell) were infected with recombinant AAV, and detected or observed by Western blotting, RT-PCR, MTT assay and flow cytometry Effects of Recombinant AAV on Cyclin B1 Expression, Proliferation, Apoptosis, and Cell Cycle of Tumor Cells in. Results rAAV-AS-CCNB1 was successfully constructed and the titer of virus was 1.0 × 1011v.g./ml. In vitro infection of U2OS cells significantly reduced its proliferation activity. Western blotting and RT-PCR suggested that rAAV-AS-CCNB1 inhibited cells Cyclin B1 expression, flow cytometry showed that the cells showed obvious apoptosis and G1 arrest. CONCLUSION: Antisense CCNB1 recombinant AAV has obvious proliferative effect on osteosarcoma U2OS cells in vitro, which may be related to the induction of apoptosis and cell cycle arrest.