目的分析不同非小细胞肺癌(NSCLC)细胞对碳离子照射放射敏感性的影响,并探讨其相关的分子机制。方法采用12C6+对肺腺癌A549细胞、鳞癌H520细胞和大细胞癌PGCL3细胞进行照射,吸收剂量分别为0、2、4和6Gy。通过克隆实验检测3种细胞系的存活率,流式细胞术检测细胞的周期分布,利用Hoechst33258染色检测细胞的凋亡率,实时RT—PCR方法检测细胞内DNA.PKcs基因的表达。结果2、4、6Gy照射后,A549、H520和PGCL3细胞的存活率明显下降,其中A549细胞辐射敏感性最低,其次是PGCL3细胞,H520细胞辐射敏感性最高。受照射后细胞均出现G2/M期阻滞与凋亡,且随剂量的升高呈现上升的趋势。受照射H520与PGCL3细胞的阻滞率(t=4.813、20.738、25.654和t=2.790、2.977,P〈0.05)和凋亡率(t=8.579、14.289、15.244和t=3.785、5.098、8.105,P〈0.05)明显高于A549细胞。受照射细胞DNA—PKcs表达明显上调,A549细胞最高,其次是PGCL3细胞,H520细胞最低;并且随着剂量的升高,DNA-PKcs的表达呈现下降趋势。2、4Gy照射后H520和PGCL3细胞DNA—PKcs的表达明显低于A549细胞(t=7.782、3.689和t=3.889、2.814,P〈0.05)。结论不同NSCLC细胞对12C6+离子的放射敏感性不同,H520鳞癌细胞最高,A549腺癌细胞最低。肺腺癌A549细胞放射敏感性较低可能与高表达的DNA—PKcs参与NHEJ途径修复DNA双链断裂有关。
Objective To study the sensitivities of different NSCLC cells irradiated with carbon ion beam and the underlying molecular mechanism. Methods Adenoearcirioma A549 cells, squamous carcinoma H520 cells and large cell carcinoma PGCL3 cells were exposed to carbon ions with different doses (0, 2, 4, 6 Gy). Cell survival fraction, apoptosis and cell cycle distribution were analyzed by colony formation assay, Hoechst33258 staining and flow eytometry assay, respectivily. DNA-PKcs mRNA expression was detected by real time RT-PCR. Results With the increasing of radiation dose, the survival of H520, PGCL3 and A549 cells were markedly reduced in order. G2/M phase arreast and apoptosis were also induced in these cells(t=4.813,20.738,25.654;t =2.790,2.977,P 〈0.05). The G2/M phase arreast rates in the irradiated H520 and PGCL3 ceils were higher than that in A549 cells. The apoptosis rates in H520 and PGCL3 ceils were also much higher than that in A549 cells (t = 8. 579, 14. 289, 15. 244; t=3.785, 5.098, 8. 105, P 〈0.05). The DNA-PKcs expression levels were upregulated in three cell lines but declined along with the increasing dose, while those in H520 and PGCL3 cells at 2 and 4 Gy were much lower than those in A549 cells (t = 7. 782, 3. 689; t = 3,889, 2. 814, P 〈0.05), Conehasions Among these three cell lines, the squamous carcinoma H520 cells have the highest radiosensitivity. Adenoeareinoma A549 cells have the lowest radiosensitivity, which may due to the high expression of DNA-PKes corresponding to NHEJ-mediated DSBs repair.