不同敏感性鼻咽癌细胞株中Ku蛋白表达情况比较

Comparison of Ku protein Expression in CNE1 and CNE2 nasopharyngeal carcinoma cell lines

目的 比较作为DNA双链断裂修复蛋白DNA依赖性蛋白激酶 (DNA PK)的调节亚基70 Ku、80 Ku在不同放射敏感性鼻咽癌细胞株CNE1和CNE2中的表达情况。方法 成克隆实验测定鼻咽癌高分化鳞癌细胞CNE1和低分化鳞癌细胞CNE2不同剂量照射后的存活分数 ,用线性二次模型拟合剂量存活曲线并求出放射生物学参数α、β、SF2、MID以评价两株细胞的放射敏感性。Westernblot检测照射前和后不同时间及不同剂量CNE1、CNE2细胞中Ku的表达 ,检测蛋白条带的吸光度值做半定量分析。结果 CNE1在各个剂量点的存活分数均比CNE2高 ,MID也大于CNE2 (2 .35∶1.11) ,SF2也大于CNE1(0 .5 4∶0 .37)。Westernblot显示两株细胞中70 Ku、80 Ku均有表达 ,蛋白条带的吸光度值测定显示70 Ku在CNE1、CNE2细胞中分别为 2 0 .0 3± 7.5 6和 19.36± 6 .0 4 ,80 Ku分别为 2 5 .98± 12 .87和 2 3.74±9.2 4 (P值均 >0 .0 5 )。蛋白表达的定量和定性分析同样显示 4Gy照射后不同时间点及 0～ 16Gy照射后 12hKu的表达无统计学差异。结论 实验验证了CNE2比CNE1对放射更敏感 ;照射前后两个细胞均有70 Ku、80 Ku蛋白表达且无差异 ;CNE1、CNE2放射敏感性的差异与Ku蛋白表达量无明显关系。

Objective Radiosensitivity is highly correlated with DNA doub le stra nd breaks(DSBs). The repair of human DSB is possible mainly through nonhomologou s DNA end joining(NHEJ)pathway. This study was designed to determine the relat ionship between expression of 70Ku/ 80Ku prot ein (a modulating subunit of DNA-PK, which is an important component in NHEJ pathway) and radiosensitivity of nasoph aryngeal carcinoma cell lines. Methods Radiosensitivity parameters of the nasoph aryngeal carcinoma cell lines were obtained by the colony forming assay and radi ation dose-survival curve was done by linear quadratic model. Western blot was p erformed to determine the expression of 70Ku and 80 Ku in total extract of CNE1 a nd CNE2 at baseline level、different time after 4?Gy irradiation、12 h after di ff erent doses of irradiation. To perform a semi-quantitated assay of protein expr e ssion, the optic density (OD) value of each band was estimated by automatic imag e analysis system. Results Surviving fraction of CNE1 was higher than those of CNE2 at each dose point. Mean inactivation dose was higher in the CNE1(2.35)t han that in the CNE2(1.11), but the quantity of 70Ku/ 80Ku in either cell line was similar at both baseline and postirradiation levels. Conclusions CNE2 is mo re radiaosensitive than CNE1; as there was no marked correlation observed betwee n the quantity of Ku protein and radiosensitivity of the nasopharyngeal carcinom a cell lines. X-irradiation cannot result in any change in total quantity of Ku protein.