DAPT对骨肉瘤U2OS细胞增殖和凋亡影响的实验研究

The effects of γ-secretase inhibitor DAPT on proliferation and apoptosis of U2OS

目的观察γ-分泌酶抑制剂DAPT对人骨肉瘤细胞系U2OS增殖和凋亡的影响,探讨DAPT抑制骨肉瘤增殖并诱导其凋亡的可能机制。方法采用体外培养人成骨肉瘤细胞系U2OS,CCK-8法检测不同浓度DAPT对成骨肉瘤细胞系U2OS生长的抑制作用;Annexin V-FITC/PI双标染色,流式细胞仪检测细胞凋亡率;Hoechst 33258染色,倒置荧光显微镜下观察细胞凋亡的形态学变化;应用RT-PCR和Western blot法检测DAPT对凋亡通路相关mRNA和蛋白表达的影响;采用Caspase活性检测试剂盒检测Caspase-3、Caspase-8、Caspase-9的活性变化。结果CCK-8检测显示5~50μmol/L的DAPT可抑制人骨肉瘤U2OS细胞增殖并具有时间和剂量依赖性;流式细胞术检测显示5、10、20μmol/L的DAPT可明显诱导骨肉瘤U2OS细胞发生凋亡,与对照组相比差异有统计学意义(P<0.05);5、10、20μmol/L的DAPT处理后,Hoechst 33258染色可见凋亡小体;RT-PCR和Western blot分析显示,经DAPT(5、10、30μmol/L)处理后的骨肉瘤细胞系U2OS表达Caspase-3、Caspase-8、Caspase-9 mRNA较对照组明显增高(P<0.05),促凋亡蛋白Bax表达增加(P<0.05),抑制凋亡蛋白Bcl-2表达减少(P<0.05);Caspase活性检测显示,DAPT(5、10、20、30μmol/L)处理后,Caspase-3、Caspase-8、Caspase-9活性较对照组明显升高(P<0.05)。结论γ-分泌酶抑制剂DAPT对骨肉瘤细胞系U2OS的增殖有显著抑制作用,并可诱导其发生凋亡,其作用机制可能与激活Caspase依赖的凋亡途径有关。

Objective To observe the effect of gamma secretase inhibitor DAPT on proliferation and apoptosis of human osteosarcoma cell line U2OS, and to explore the possible mechanism of DAPT inhibiting osteosarcoma proliferation and inducing apoptosis. Methods Osteosarcoma cell line U2OS was cultured in vitro. Cell proliferation was tested by cell counting kit-8 （CCK-8） assay. Double stained by Annexin V-FITC and Propidium Iodide （PI）, the cells were detected by flow cytometry （FCM） for apoptosis. Hoechst 33258 was used to detect the morphological change of typical apoptotic cells. The apoptosis related mRNA and protein levels in U2OS were detected by RT-qPCR and Western blot. The activities of Caspase-3, Caspase-8 and Caspase-9 were measured with Caspase activity assay kit. Results CCK-8 assay showed that 5-50 μmol/L DAPT inhibited the proliferation of human osteosarcoma U2OS cells and in a time- and dose- dependent manners. Flow cytometry showed that 5, 10 and 20 μmol/L DAPT could significantly induce apoptosis of osteosareoma U2OS cells, compared to the control group, the differences were statistically significant （P 〈 0.05）. Apoptotic body could be observed by Hoechst 33258 staining after treatment with 5, 10 and 20 μmol/L DAPT. RT-PCR and Western blot analysis showed that after treatment with DAPT （5, 10, 30 μmol/L）, the expression of Caspase- 3, 8,9 mRNA were higher than those of control group （P 〈 0.05）, the pro-apoptotic protein Bax increased （P 〈 0.05）, and anti-a, poptosis protein Bcl-2 protein decreased （P 〈 0.05）. The activities of Caspase-3, Caspase-8 and Caspase-9 promoted obviously after treatment with DAPT （5, 10, 20, 30 μmol/L） when compared with the control group （P 〈 0.05）. Conclusion DAPT can significantly inhibit the proliferation and induce apoptosis of human osteosarcoma cell line U20S. Its mechanism of action may be related to activation of Caspase dependent apoptosis pathway.