pSilence APE1联合中子射线治疗骨肉瘤的体外实验研究

In vitro study of the treatment of human osteosarcoma by pSilence APE1 combined with neutron-ray

目的应用双功能基因脱嘌呤/脱嘧啶核酸内切酶(APE1)小干扰RNA(siRNA)表达载体pSilence APE1“敲低”人骨肉瘤HOS细胞APE1的表达,观察pSilence APE1联合中子放射治疗对HOS细胞生长抑制的协同作用,并探讨其可能机制。方法用脂质体将pSilence APE1质粒导入HOS细胞,同时给予不同剂量的锎-252中子射线照射,采用克隆形成分析法测算平均致死剂量(D0)、准域剂量(Dq)值,彗星分析法检测照射后细胞的DNA损伤情况,并用流式细胞仪检测细胞凋亡情况。结果克隆形成分析显示对照组与pSilence APE1转染组HOS细胞经锎-252中子射线照射后的D0值分别为2·80和1·89,Dq值分别为2·66和2·00。分别以2、5、10Gy锎-252中子射线照射对照组与pSilence APE1转染组HOS细胞后,碱性彗星尾力矩分别为6·664±0·648、20·322±1·433、33·909±1·245和7·997±0·542、25·238±1·185、39·191±1·052(P<0·01);细胞凋亡率分别为4·00%、5·91%、9·63%和5·68%、7·55%、13·51%(P<0·05)。结论pSilenceAPE1能显著提高HOS细胞对中子射线的敏感性,促进DNA损伤和细胞凋亡。pSilence APE1基因联合中子放疗可能成为今后骨肉瘤治疗的新方法。

Objective Apuinic/apyrimidic endonuclease/redox effector factor-1 （APE1/Ref-1, abbreviated as APE1） is a major member of the base excision repair （BER） pathway involved in oxidative DNA damage repair. To knock down APE1 gene expression in HOS cells with pSilence APE1, and explore its effect in combination with 252^Cf neutron ray radiotherapy. Methods The constructed APE1 siRNA expression vector pSilence APE1 was transfected into HOS cells by SuperFect Transfection liposome, and it was used to knock down the expression of APE1. The HOS cells and transfected HOS cells were respectively irradiated by 252^Cf neutron ray, then the cell survival （D0）, DNA single strand breaks （SSB） and cell apoptosis were determined by clone formation assay, alkaline comet assay and flow cytometer. Results The cell-survival curve was plotted by clone formation assay, the D0 value was 2. 80 vs. 1.89 and Dq value was 2. 66 vs. 2. 00 for the control and transfected HOS cells, respectively, after being irradiated by 252^Cf neutron ray. The tail moments at 2, 5 and 10 Gy were 6. 664±0. 648 vs. 7. 997±0. 542, 20. 322±1.433 vs. 25. 238±1. 185 and 33. 909±1. 245 vs. 39. 191±1.052, respectively, for the control and transfected HOS cells, and the cell apoptosis rate at 2, 5 and 10 Gy was 4. 00 w 5. 68, 5. 91 vs. 7. 55 and 9.63 vs. 13. 51, respectively, for the control and transfected HOS cells. All these findings showed significant difference between the two groups （P〈 0.05）. Conclusion The results indicated that pSilence APE1 may significantly enhance the sensitivity of HOS cells to neutron radiotherapy, and hasten DNA damage and cell apoptosis. Therefore, the gene therapy with pSilence APE1 combined 252^Cf neutron radiotherapy may be a promising approach to treat human osteosarcoma in the future.