端粒酶阴性肿瘤细胞株U20S中Ku80表达抑制模型的建立及其对放射敏感性的影响

The Ku80 inhibition cell model in telomerase-negative tumor cell lines U2OS and its relation to telomere and radiosensitivity

目的探讨Ku80在端粒酶阴性肿瘤细胞中与端粒和放射敏感性的关系。方法构建重组表达质粒pshRNA—Ku80，转染U20S细胞，并筛选稳定表达的转化克隆。RT—PCR和Westernblot分别在基因和蛋白水平检测干扰前后Ku80表达量的变化。定时PCR检测干扰前后端粒长度的变化。克隆形成实验分析pshRNA—Ku80干扰对U20S细胞放射敏感性的影响。结果流式细胞仪检测重组质粒稳定转染细胞株的转染效率为（83．23±7．63）％。PCR结果显示，重组质粒组Ku80基因抑制率为（68．09±1．16）％。Westernblot结果表明，重组质粒组Ku80蛋白抑制率达到（11．03±2．45）％。端粒长度检测结果显示，重组质粒组的端粒长度（1．07±0．07）明显短于空白组（4．42±1．30）（F=38．58，P〈0．05），而空质粒组端粒长度（4．1l±0．84）与空白组无明显变化。克隆形成实验结果显示，重组质粒组细胞株SF。值较空白组降低显著（F=1089．61，P〈0．05），放射增敏比（SER）为1．47。结论运用RNAi技术所建立的Ku80表达抑制的细胞模型，可用于以后的基因功能研究；在U20S中，特异性地沉默Ku80会引起端粒长度的缩短，并增加U20S细胞的放射敏感性，推测pshRNA—Ku80引起端粒缩短很可能是其放射增敏的机制之一。

Objective To construct the KUS0 inhibition cell model by RNAi in U2OS cell and to explore the relationship between the Ku80, telomeres and radiosensitivity in telomerase-negative tumor ceils. Methods U2OS cells were transfected with the recombinant plasmids of pshRNA-K80 by the lipofectamine, and the stable transfected cell clones were selected by G418. After the selection, the cells were collected and analyzed by the flow cytometry. RT-PCR and Western blot were used to measure the expression of Ku80 and Real-time PCR was used to detect the length of telomeres. The radiosensitivity of U2OS was determined by clone formation array. Results The transfection efficiency of the positive cell clones detected by the flow cytometry was （83.23 ± 7.63） %. The inhibition rate of the Ku80 gene transcription in the cell group with recombinant plasmid was（68.09 ± 1.16）% and the inhibition rate of the Ku80 protein expression in the same group was （ 11.03 ± 2.45 ） %. The results of Real-time PCR showed that the telomere length of the cell group with recombinant plasmid （ 1.07 ±0. 07） was significantly shorter than that of the control group （4.42 ±1.30, F = 38.58, P 〈 0.05） and that of the empty plasmid group （4. 11 ±0.84, F = 38.58, P 〈 0. 05 ）. Compared to the control group, the telomere length of the empty plasmid group did not changed（4. 42 ±0. 84 vs. 4. 11 ±0.84）. U2OS cells with Ku80 expression suppressed had lower SF2 than that of the control cells （F = 1089.61, P 〈 0. 05） , and resulted in the SER of 1.47. Conclusions The Ku80 inhibition cell model in telomerase-negative U2OS cell line is successfully established which has the shorter telomere length, and is more sensitive to radiation. Telomereshortening caused by pshRNA-of KuSO is likely to be one of the mechanisms of radiosensitization in this kind of cell model.