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本文土要研究DNA依赖的蛋白激酶(DNA-dependent protein kinase,DNA-PK)与鼻咽癌细胞放射敏感性之间的关系。克隆形成实验分析鼻咽癌细胞CNE1/CNE2的剂量存活曲线,Signa TECT DNA-PK试剂盒检测DNA-PK活性,免疫荧光及激光显微共聚焦分析放疗前及放疗后15min、1h、6h、12h和24h CNE1/CNE2细胞中Kus及DNA-PKcs的亚细胞定位,Western blot分析两株细胞中Kus蛋白的表达。结果显示:CNE1细胞在每个剂量点的存活分数均高于CNE2细胞;同时发现放疗前后CNE1细胞中的DNA-PK活性也均高于CNE2细胞,但两株细胞中Ku70/Ku80蛋白表达无明显差异;放疗可使DNA- PK活性增加,且各个检测时间点CNE1细胞增加的幅度大于CNE2细胞;DNA-PK亚基可同时定位于胞浆和胞核,但主要位于胞核,细胞照射后Ku70、Ku80和DNA-PKcs从胞浆转运到胞核。结果表明:DNA-PK活性更高可能足CNE1细胞较CNE2细胞更能抵抗放射的原因之一;放疗所致DNA-PK活性增高可能与DNA-PK亚基从胞浆转运到胞核有关,而与Ku蛋白表达的总量无关。
In this study, we investigated the relationship between DNA-dependent protein kinase (DNA-PK) and radiosensitivity of nasopharyngeal carcinoma cells. The survival curves of CNE1 / CNE2 cells in nasopharyngeal carcinoma cells were analyzed by clonogenic assay. The DNA-PK activity was detected by Signa TECT DNA-PK kit. Immunofluorescence and laser scanning confocal microscopy were used to analyze the dose-dependent survival curves at 15 min, 1 h, 6 h, 12 h And subcellular localization of Kus and DNA-PKcs in 24h CNE1 / CNE2 cells. The expression of Kus protein in the two cell lines was analyzed by Western blot. The results showed that the survival score of CNE1 cells at each dose point was higher than that of CNE2 cells. The DNA-PK activity of CNE1 cells before and after radiotherapy was also higher than that of CNE2 cells, but the expression of Ku70 / Ku80 protein was not significant DNA-PK subunit can be located in the cytoplasm and nucleus at the same time, but mainly located in the nucleus. After the cells were irradiated, the activity of Ku-70 was significantly higher than that of CNE2 cells , Ku80 and DNA-PKcs are transported from the cytoplasm to the nucleus. The results showed that the higher DNA-PK activity may be one of the reasons that CNE1 cells are more resistant to radiation than CNE2 cells. The increased DNA-PK activity may be related to DNA-PK subunit transport from the cytoplasm to the nucleus, Not related to the total amount of Ku protein expression.