鼻咽癌放射抵抗细胞株的建立与鉴定

Establishment and identification of radioresistant nasopharyngeal carcinoma cell line

目的体外构建鼻咽癌放射抵抗细胞株, 为进一步研究鼻咽癌放射抵抗的分子机制提供实验基础。方法采用剂量梯度法照射诱导建立放射抵抗细胞模型5-8F-IR。光学显微镜观察细胞形态变化;克隆形成实验检测细胞克隆形成能力;CCK-8实验检测细胞活力;5-溴-2-脱氧尿嘧啶(EdU)实验检测细胞增殖能力;彗星实验及免疫荧光实验检测细胞DNA损伤修复能力;流式细胞术检测细胞凋亡水平及周期分布;蛋白质印迹法检测DNA损伤相关蛋白γH2AX及凋亡相关蛋白胱天蛋白酶3(Caspase-3)的蛋白表达水平。结果剂量梯度照射后5-8F-IR细胞形态较亲本5-8F细胞形态变长, 5-8F-IR细胞的克隆形成能力(P<0.01)、细胞活力(P<0.001)、细胞增殖能力(P<0.05)、DNA损伤修复能力(P<0.05)均明显优于亲本细胞5-8F, 照射后5-8F-IR细胞的凋亡率明显低于5-8F细胞(P<0.01), 未接受照射时5-8F-IR细胞处于S期的百分比较5-8F细胞升高, 接受照射后5-8F-IR细胞出现明显的G_(2)/M期阻滞(P<0.01), 同时照射后5-8F-IR细胞的DNA损伤相关蛋白γH2AX(P<0.001)及凋亡相关蛋白Caspase-3(P<0.05)蛋白表达水平明显低于5-8F细胞。结论人鼻咽癌细胞株5-8F经剂量梯度法照射诱导建立的5-8F-IR细胞具有放射抗拒性并显示出与亲代5-8F细胞不同的生物学特性, 为探索鼻咽癌放射抵抗机制提供了研究工具。

Objective To establish a radioresistant nasopharyngeal carcinoma cell line in vitro and provide experimental basis for further research of the molecular mechanism of radioresistance.Methods A radioresistant cell line 5-8F-IR was established by dose gradient irradiation.Cell morphological changes were observed by optical microscope.The formation of colony was detected by colony formation assay.Cell viability was detected by CCK-8 assay.Cell proliferation was assessed by 5-ethynyl-2'-deoxyuridine(EdU)assay.DNA damage repair ability was measured by immunofluorescence staining and comet assay.Cell apoptosis and cycle were detected by flow cytometry.The expression levels of DNA damage related protein H2AX and apoptosis related protein Caspase-3 were measured by Western blot.Results The 5-8F-IR cells became longer than that of parental generation 5-8F cells after dose gradient irradiation.The colony formation ability(P<0.01),cell viability(P<0.001),cell proliferation ability(P<0.05)and DNA damage repair ability(P<0.05)of 5-8F-IR cells were significantly stronger than those of parental generation 5-8F cells.The apoptosis rate of 5-8F-IR cells after irradiation was significantly lower than that of parental generation 5-8F cells(P<0.01).The 5-8F-IR cells showed higher percentage of cells in S phase without irradiation,and obvious G_(2)/M phase arrest after irradiation(P<0.01).Western blot showed that the expression levels of H2AX(P<0.001)and Caspase-3(P<0.05)in 5-8FIR cells after irradiation were significantly lower compared with those of parental generation 5-8F cells.Conclusion 5-8F-IR cells induced by dose gradient irradiation of human nasopharyngeal carcinoma cell line 5-8F cells exhibit radioresistance and exhibit different biological characteristics compared to their parental 5-8F cells,providing a research tool for exploring radioresistance mechanism of nasopharyngeal carcinoma.