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目的探讨p14ARF对顺铂诱导骨肉瘤U2OS细胞凋亡的影响及其机制,为提高骨肉瘤化疗敏感性提供依据。方法在不表达p14ARF的U2OS细胞中稳定转染pcDNA3.1-p14ARF质粒,构建稳定表达株;采用RT-PCR和Western blot法鉴定其表达;台盼蓝拒染法测定生长抑制作用;Hoechst33258荧光染色检测凋亡;Western blot法检测Caspase-3,9和PARP的表达和活化。结果 mRNA和蛋白水平,U2OS和U2OS-vec细胞未见p14ARF表达,U2OS-ARF细胞可见p14ARF的高表达;p14ARF单独对U2OS细胞的生长无影响,但顺铂(5μM)处理72小时后,U2OS-ARF细胞的生长抑制率明显高于U2OS和U2OS-vec细胞,相对于U2OS和U2OS-vec细胞,U2OS-ARF细胞不仅表现更为明显的凋亡形态学变化,还明显出现了Caspase-3,9和PARP的活化裂解。结论 p14ARF能够增强顺铂诱导的骨肉瘤U2OS细胞毒作用和凋亡,通过激活Caspase-PARP级联活化,提高骨肉瘤U2OS细胞对顺铂的敏感性。
Objective To investigate the effect and mechanism of p14ARF on cisplatin-induced apoptosis of osteosarcoma U2OS cells and provide basis for improving the chemosensitivity of osteosarcoma. Methods The recombinant plasmid pcDNA3.1-p14ARF was successfully transfected into U2OS cells without p14ARF expression. The stable expression strain was constructed by RT-PCR and Western blot. The growth inhibition was determined by trypan blue exclusion assay. The expression of Hoechst33258 Apoptosis was detected. Western blot was used to detect the expression and activation of Caspase-3, 9 and PARP. Results The mRNA and protein levels of p14ARF were not found in U2OS and U2OS-vec cells, while the expression of p14ARF was found in U2OS-ARF cells. However, p14ARF alone had no effect on the growth of U2OS cells. After 72 hours of cisplatin treatment, U2OS- ARF cell growth inhibition rate was significantly higher than that of U2OS and U2OS-vec cells, compared with U2OS and U2OS-vec cells, U2OS-ARF cells not only showed more obvious morphological changes of apoptosis, but also obviously appeared Caspase-3,9 And activated cleavage of PARP. Conclusion p14ARF can enhance cisplatin-induced osteosarcoma U2OS cytotoxicity and apoptosis, and increase the sensitivity of U2OS cells to cisplatin by activating Caspase-PARP cascade activation.