目的：探讨环氧化酶￣2(COX￣2)抑制药塞来昔布对鼻咽癌放射抗拒细胞株 CNE￣2R 放射敏感性的影响及其机制。方法人鼻咽癌 CNE￣2细胞株经2,4,6,8 Gy 梯度剂量照射,每个剂量照射3次,构建鼻咽癌放射抗拒亚株 CNE￣2R,克隆形成实验验证其放射敏感性,分别给予25,50,75μmol.L－1塞来昔布作用于 CNE￣2及 CNE￣2R 细胞后,Western blotting实验检测 COX￣2蛋白表达变化,克隆形成实验检测单纯放疗或放疗联合30μmol.L－1塞来昔布作用于 CNE￣2和 CNE￣2R细胞后的存活分数差异,流式细胞术检测单纯放疗或放疗联合塞来昔布对细胞凋亡的影响,免疫荧光实验检测放疗后鼻咽癌细胞核内γ￣H2AX 荧光焦点数量。结果放射抗拒 CNE￣2R 细胞放射生物学参数 SF2、D0、Dq 及 N 值分别为(0.81±0.05),(2.15±0.07) Gy,(2.94±0.08) Gy,(3.91±0.07),均显著高于 CNE￣2细胞株(0.61±0.08),(1.47±0.06) Gy,(1.68±0.10) Gy,(3.13±0.05)；塞来昔布预处理后 COX￣2蛋白表达随药物浓度增高而显著下调；克隆形成实验显示塞来昔布作用后, CNE￣2及 CNE￣2R 细胞经射线照射后存活分数均降低；射线照射联合塞来昔布作用于 CNE￣2细胞后其凋亡率[(13.10±0.63)％]较对照组[(4.90±0.71)％]增加,作用于 CNE￣2R 细胞后凋亡率[(5.30±0.75)％]较对照组[(1.82±0.82)％]增加；塞来昔布处理后诱导鼻咽癌细胞株的放射敏感性增高,射线照射24 h 后细胞核内γ￣H2AX 荧光焦点数增加,两两比较差异均有统计学意义(P<0.05)。结论塞来昔布能够增强鼻咽癌放射抗拒细胞株 CNE￣2R 的放射敏感性。%Objective To investigate the effect of COX￣2 inhibitor celecoxib on radiosensitity of irradiation￣resistant cell line CNE￣2R of nasopharyngeal carcinoma and the potential mechanism. Methods Via exposing to a series of X￣ray (2, 4, 6, 8 Gy, 3 times for each dose), radio￣resistant cell subline CNE￣2R was established from human nasopharyngeal carcinoma cell CNE￣2.Radiosensitivity was detected by clone formation assay.CNE￣2R and CNE￣2 cell lines were exposed to 25, 50, 75 μmol.L-1 celecoxib, respectively.Western blotting was used to detect the protein expression of COX￣2.Clone formation assay was performed to measure the survival fraction of CNE￣2 and CNE￣2R after radiotherapy alone or radiotherapy combined with 30 μmol.L-1 celecoxib treatment.Flow cytometry was used to measure influence of radiotherapy alone or radiotherapy combined with 30 μmol.L￣1celecoxib treatment on cell apoptosis.Number of residual γ￣H2AX foci was observed by immunofluorescence assay. Results The colony forming assay demonstrated that the values of SF2, D0 , Dq , and N of CNE￣2R cell subline [(0.81±0.05), (2.15±0.07) Gy, (2.94±0.08) Gy, (3.91±0.07), respectively] was significant higher than those of CNE￣2 cell line [(0.61±0.08), (1.47±0.06) Gy, (1. 68 ± 0. 10) Gy, (3. 13 ± 0. 05), respectively]. The expression of COX￣2 protein was significantly downregulated with increasing celecoxib concentration.Surviving fraction was decreased in both CNE￣2 and CNE￣2R cell lines after irradiation.After radiotherapy combined with celecoxib, apoptosis rates of CNE￣2 and CNE￣2R cell lines [(13.10±0.63)%, (5.30±0.75)%] were higher than those of the corresponding control groups [(4.90±0.71)%, (1.82±0.82)%].Celecoxib increased radiosensitivity in nasopharyngeal carcinoma CNE￣2R and CNE￣2 cell lines.The number of residual γ￣H2AX foci after irradiation was increased by celecoxib pretreatment.The difference was statistically significant (P<0.05). Conclusion Celecoxib can enhance radiosensitivity of radio￣resistant cell subline CNE￣2R of human nasopharyngeal carcinoma in vitro.
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