摘要
目的观察耐替莫唑胺人脑胶质瘤U251细胞（U251／TR）经放射线照射后多药耐药基因（MDR1）及P-糖蛋白（P-gp）表达的改变。方法体外常规培养U251细胞，低浓度替莫唑胺分步诱导法诱导建立耐药细胞U251／TR。细胞活性检测试剂盒（CCK-8）检测替莫唑胺、阿霉素、依托泊苷、硫酸长春新碱和环磷酰胺对U251、U251／TR细胞的相对抑制率和耐药指数（RI）。采用双光子医用直线加速器产生的不同剂量（2、4、6、8、10、12、15Gv）X线照射U251、U251／TR细胞24h后，RT—PCR检测细胞MDR1的表达，Westem blotting检测细胞膜P—gp的表达。结果CCK-8检测结果显示替莫唑胺、阿霉素、环磷酰胺、依托泊苷、硫酸长春新碱对U251细胞的抑制率分别为（19．3±0．04）％、（39．9±0．13）％、（28．1±0．09）％、（66．2±0．02）％、（26.3±0．11）％，对U251／TR细胞的抑制率分别为（3．2±0．03）％、（15.3±0．09）％、（4．1±0．03）％、（45．6±0．10）％、（10．8±0．04）％，U251/TR细胞对上述5种药物的RI分别为U251细胞的2．63、2．76、2．15、10．58、3．54倍；RT—PCR检测显示：2Gy、4Gy、6Gy放射线照射U251／TR和U251细胞后，2种细胞的MDR1基因表达均依次下降。且6Gy时降至最低。但经大剂量如8Gy、10Gy、12ay、15GyX线照射后，2种细胞的MDR1基因表达开始依次上升，均高于6Gy照射组。Western blotting结果显示：6Gy放射线照射U251／TR和U251细胞后，P—gp的蛋白表达均下降。结论小剂量的X线（8ay以下）照射后U251、U251／TR细胞的放射敏感性均增高，但大剂量的X线（8Gy以上）照射后U251／TR、U251细胞均产生放射抗拒。
Objective To explore the expression changes ofmultidrug-resistant gene （MDR1） and P-glycoprotein （P-gp） in temozolomide （TMZ） resistant U251 cell lines （U251/TR） after radiation exposure. Methods U251 cells were routinely cultured in vitro; low concentration oftemozolomide was added to the cells to induce TMZ resistant U251 cell lines （U251/TR）; CCK-8 method was employed to assay the relative inhibition rate and resistance index （RD of U251 and U251/TR to multi-drugs （TMZ, adriamycin, etoposide, vincristine sulfate and cyclophosphamide）; U251 parents and U251/TR cells were exposed to X-rays of different doses （2, 4, 6, 8, 10, 12 and 15 Gy） by linear accelerator for 24 h, and the MDR1 mRNA expressions in the two cells lines were assayed by reverse transcription-PCR, and P-gp protein expressions were detected by Western blotting. Results CCK-8 method indicated that the relative inhibition rate of U251 to TMZ, adriamycin, etoposide, vincristine sulfate and cyclophosphamide was （19.3±0.04）%, （39.9±0.13）%, （28.1±0.09）%, （66.2±0.02）% and （26.3±0.11）%, respectively, and that of U251/TR to the above drugs was （3.2±0.03）%, （15.3±0.09）%, （4.1±0.03）%, （45.6±0.10）% and （10.8±0.04）%;RI of U251/TR to TMZ, adriamycin, etoposide, vincristine sulfate and cyclophosphamide was 2.63, 2.76, 2.15, 10.58 and 3.54 times of that of U251. Reverse transcription-PCR indicated that after 2, 4 and 6 Gy X-rays exposure, MDR1 gene expressions in the two cell lines were both gradually deceased, reaching the lowest level at 6 Gy; however, after 8, 10, 12 and 15 Gy X-rays exposure, MDR1 gene expressions in the two cell lines were both gradually increased. Western blotting showed that the P-gp protein expression was decreased after 6 Gy X-ray irradiation exposure. Conclusion The radiosensitivity in both U251 and U251/TR cells can be increased after small doses X-ray （such as 6 Gy） radiation exposure, while the high-dose （8 Gy above） irradiation can induce radiation-resistance in U251/TR and U251 cells.
出处
《中华神经医学杂志》
CAS
CSCD
北大核心
2016年第1期60-64,共5页
Chinese Journal of Neuromedicine
基金
国家自然科学基金（30973072）
