中性彗星分析法检测肿瘤细胞的放射敏感性

Neutral Comet Assay in Detecting the Intrinsic Radiosensitivity of Tumor Cells

[目的]研究中性彗星分析法检测肿瘤细胞内在放射敏感性的可靠性和影响因素。[方法]人鼻咽高分化鳞癌细胞株CNE鄄1和人纤维肉瘤细胞株HT1080细胞,用6MVX线以0、100、200、300、400、600、800、1000cGy等不同剂量点照射,克隆形成法制定存活曲线,比较其放射敏感性的差异。然后进行两种细胞于600cGy照射后1、2、4、8h的中性彗星分析,以尾力矩为指标,动态检测不同时间点残留的DNA双链断裂,并与克隆形成分析结果相比较。[结果]克隆形成分析法显示CNE鄄1细胞的放射敏感性低于HT1080细胞,其2Gy照射后存活分数(SF2)和平均致死剂量D0值分别为0.397、0.331和1.898、1.287。两种细胞在照射后1、2、4h的中性彗星尾力矩分别为0.148±0.106、0.100±0.083、0.058±0.043和0.297±0.179、0.157±0.092、0.092±0.060,各时间点之间有显著性差异(P<0.05),但差别逐渐减小;照射后2h和4h的尾力矩比值分别为0.637和0.630,与SF2比值和D0比值的倒数(0.834和0.678)较接近;两种细胞照射后8h的尾力矩比较差异无显著性。[结论]中性彗星分析法与克隆形成分析法的检测结果有确切的相关性,照射后一定时间内的残留DNA双链断裂有可能成为检测肿瘤细胞放射敏感性的指标。

Purpose]To investigate the reliability and the influencing factors of neutral comet assay in detecting the intrinsic radiosensitivity of tumor cells.[Methods]Two human cell lines (differentiated nasopharyngeal squamous carcinoma CNE-1 and fibrosarcoma HT1080) were irradiated with 6MV X-ray in different dose points of 0cGy,100cGy,200cGy, 300cGy, 400cGy, 600cGy, 800cGy, and 1000cGy, Intrinsic radiosensitivity of the two cells was compared according to the survival curves processed by clonogenic assay.Then the tumor cells irradiated to a dose of 600cGy underwent neutral comet assay at 1h, 2h, 4h, and 8h after irradiation, and their residue DNA double strands breaks (DSBs) at these time points were determined respectively with the tail moment,and compared with the results of clonogenic assay.[Results]The results of clonogenic assay suggested that the radiosensitivity of CNE-1 cell was lower than that of HT1080 cell with the survival fractions at 2Gy(SF2, 0.397 vs. 0.331) and mean lethal dose(D0) values(1.898 vs. 1.287) respectively. In the neutral comet assay, the tail moments of CNE-1 and HT1080 cell lines at 1h, 2h and 4h after irradiation were 0.148±0.106, 0.100±0.083, 0.058±0.043 and 0.297±0.179,0.157±0.092,0.092±0.060, respectively.The tail moments at each time point decreased gradually with significant difference (P<0.05). The ratio of tail moments at 2h and 4h (0.637 and 0.630) was closed to the inverse ratios of SF2 and D0(0.834 and 0.678).The tail moments of the two cell lines showed no significant difference at 8h after radiation.[Conclusion]The results of neutral comet assay can be reliably related to the radiosensitivity measured with clonogenic assay,the amount of residual DNA DSBs within a given time after irradiation may be a valid parameter for radiosensitivity prediction in tumor cells.