小白菊内酯对骨肉瘤细胞凋亡与凋亡相关蛋白的影响及作用机制研究

Effect and mechanism of parthenolide on apoptosis and apoptosis-related proteins of osteosarcoma cells

目的:探讨小白菊内酯(PTL)对骨肉瘤细胞凋亡及凋亡相关蛋白的影响及作用机制。方法:CCK-8实验检测并筛选出PTL抑制骨肉瘤细胞活性的有效作用浓度,再通过流式细胞法检测PTL促进骨肉瘤细胞发生凋亡的有效作用浓度,最终确定出用于后续实验的有效PTL浓度,实时荧光定量PCR法和Western blot法从分子水平检测PTL对骨肉瘤细胞凋亡及相关凋亡蛋白表达的影响。结果:CCK-8实验结果表明,PTL浓度为5、10μmol/L时,对骨肉瘤细胞活力和凋亡均无显著影响。而PTL浓度为20、40μmol/L时,显著抑制骨肉瘤细胞活性(均P<0.05),PTL浓度为20、40μmol/L时,显著促进骨肉瘤细胞发生凋亡,且20、40μmol/L PTL两组间比较差异无统计学意义(均P>0.05)。故选择20μmol/L PTL用于后续实验,细胞爬片及TUNEL实验证实PTL(20μmol/L)显著促进骨肉瘤细胞凋亡(P<0.05),实时荧光定量PCR法和Western blot法表明PTL显著促进骨肉瘤细胞Bax mRNA和蛋白表达上调,Cleaved-Caspase 3/Caspase 3比值上调,并抑制Bcl-2 mRNA和蛋白表达(均P<0.05)。结论:PTL通过上调Bax表达及Cleaved-Caspase 3/Caspase 3比值,并抑制Bcl-2表达,进而体外诱导骨肉瘤细胞发生凋亡。

Objective:To detect the effect and mechanism of parthenolide(PTL)on apoptosis and apoptosis-related proteins of osteosarcoma cells in vitro.Methods:CCK-8 experiment detected and screened the effective concentration of PTL to inhibit the activity of osteosarcoma cells,and then detected the effective concentration of PTL to promote osteosarcoma cell apoptosis by flow cytometry,and finally determined the effective concentration for subsequent experiments.Real-time fluorescent quantitative PCR and Western blot methods were used to detect the influence of PTL on osteosarcoma cell apoptosis and the expression of related apoptotic proteins at the molecular level.Results:The results of the CCK-8 experiment showed that the PTL at concentration of 5 and 10μmol/L had no significant effect on the viability and apoptosis of osteosarcoma cells.PTL at concentrations of 20 and 40μmol/L significantly inhibited the activity of osteosarcoma cells(all P<0.05),and significantly promoted osteosarcoma cell apoptosis(all P<0.05),but there was no statistically significant difference between 20μmol/L PTL and 40μmol/L PTL(P>0.05).Therefore,20μmol/L PTL was selected for follow-up experiments.Cell slide and TUNEL experiments confirmed that PTL(20μmol/L)significantly promoted osteosarcoma cell apoptosis(P<0.05).Real-time quantitative PCR and Western blot methods showed that PTL significantly promoted the up-regulation of Bax mRNA and protein expression in osteosarcoma cells,and up-regulated the ratio of Cleaved-Caspase 3 to Caspase 3,and inhibited the expression of Bcl-2 mRNA and protein(all P<0.05).Conclusion:PTL up-regulates the expression of Bax and the ratio of Cleaved-Caspase 3 to Caspase 3,and inhibits the expression of Bcl-2,thereby inducing osteosarcoma cell apoptosis in vitro.