CAJ | 학술논문

目的体外实验探讨放射线照射对鼻咽癌细胞上皮-间质转化的影响.方法采用梯度递增放射线照射筛选构建鼻咽癌CNE-2放疗抵抗细胞(CNE-2 with radio resistance,CNE-2-Rs),并通过CCK-8法、克隆形成实验、流式细胞术验证CNE-2-Rs细胞的放疗抵抗性.显微镜下观察照射过程中,CNE-2细胞的形态学改变；Western blot检测不同照射时期CNE-2细胞上皮细胞标志物钙黏蛋白和间质细胞标志物波形蛋白的表达情况.结果 CCK-8结果表明,CNE-2-Rs细胞在同一时间点(第4天)不同照射剂量(4、6、8 Gy)以及同一照射剂量(4 Gy)不同时间点(2～5 d)下的存活率均高于其亲本细胞(P值均＜0.05)；4 Gy放射线照射前细胞增殖能力基本一致,放射线照射后CNE-2增殖能力明显低于CNE-2-Rs；CNE-2-Rs细胞放射线处理后,集落形成能力强于亲本细胞(P值均＜0.05)；拟合多靶单击模型和线性二次模型细胞存活曲线,结果表明,平均致死剂量(mean lethal dose,D0)、准阈剂量(quasi-threshold dose,Dq)、2 Gy细胞存活分数(survival fraction,SF2)和平均灭活剂量(mean inactivation dose,MID)增加,α和α/β值减少(P值均＜0.05)；CNE-2-Rs细胞接受4Gy射线后,相对于CNE-2细胞出现S期、G2期细胞比例增多(P值均＜0.05).CNE-2-Rs细胞在形态上出现呈梭形、伪足伸出和细胞间连接变松散等上皮-间质转化形态学改变.照射总剂量达24 Gy后,CNE-2细胞钙黏蛋白表达下调和波形蛋白表达升高(P值均＜0.01).结论鼻咽癌细胞在放射线照射过程中出现典型的上皮-间质转化形态学及分子标志物改变,提示上皮-间质转化可能在鼻咽癌放疗抵抗中发挥了重要作用.
목적 체외실험탐토방사선조사대비인암세포상피-간질전화적영향.방법 채용제도체증방사선조사사선구건비인암CNE-2방료저항세포(CNE-2 with radio resistance,CNE-2-Rs),병통과CCK-8법、극륭형성실험、류식세포술험증CNE-2-Rs세포적방료저항성.현미경하관찰조사과정중,CNE-2세포적형태학개변；Western blot검측불동조사시기CNE-2세포상피세포표지물개점단백화간질세포표지물파형단백적표체정황.결과 CCK-8결과표명,CNE-2-Rs세포재동일시간점(제4천)불동조사제량(4、6、8 Gy)이급동일조사제량(4 Gy)불동시간점(2～5 d)하적존활솔균고우기친본세포(P치균＜0.05)；4 Gy방사선조사전세포증식능력기본일치,방사선조사후CNE-2증식능력명현저우CNE-2-Rs；CNE-2-Rs세포방사선처리후,집락형성능력강우친본세포(P치균＜0.05)；의합다파단격모형화선성이차모형세포존활곡선,결과표명,평균치사제량(mean lethal dose,D0)、준역제량(quasi-threshold dose,Dq)、2 Gy세포존활분수(survival fraction,SF2)화평균멸활제량(mean inactivation dose,MID)증가,α화α/β치감소(P치균＜0.05)；CNE-2-Rs세포접수4Gy사선후,상대우CNE-2세포출현S기、G2기세포비례증다(P치균＜0.05).CNE-2-Rs세포재형태상출현정사형、위족신출화세포간련접변송산등상피-간질전화형태학개변.조사총제량체24 Gy후,CNE-2세포개점단백표체하조화파형단백표체승고(P치균＜0.01).결론 비인암세포재방사선조사과정중출현전형적상피-간질전화형태학급분자표지물개변,제시상피-간질전화가능재비인암방료저항중발휘료중요작용.
Objective To investigate the irradiation induced epithelial-mesenchymal transition (EMT) in nasopharyngeal carcinoma (NPC) in vitro.Methods NPC CNE-2 cells with radioresistance (CNE-2-Rs) were established by exposure to gradiently increased dose of irradiation.CCK-8 cell viability kits,colony formation assay and fluorescence-activated cell sorting analysis were used to confirm the capacity of radioresistance of CNE-2-Rs cells.Invert microscope was used to monitor the morphological changes and western blot was applied to detect the expression of epithelial cell marker E-cadherin and mesenchymal cell marker Vimentin during the phase of CNE-2 exposure to irradiation.Results Irradiation exposure successfully induced the radioresistance of CNE-2 cells.After exposed to irradiation,the survival rate in CNE-2-Rs was higher than that in CNE-2 by CCK-8 assays.No significant difference of proliferation ability was observed between the CNE-2 and CNE-2-Rs pre-radiotherapy,but a higher proliferation ability in the CNE-2-Rs post-radiotherapy.By using the colony forming assay,the parameters of CNE-2 and CNE-2-Rs in multi-target single-hit and linear quadratic model were obtained and the data demonstrated that parameters mean lethal dose (D0),quasi-thres hold dose (Dq),surrival fraction in 2Qy (SF2) and mean inctivation dose (MID) value increased,α and α/β value decreased(P ＜0.05).At the same time,the CNE-2-Rs cells showed higher percentage of cells in S and G2 phase(P ＜ 0.05).In terms of biomorphology,CNE-2-Rs cells were more narrow,long strips or fusiform shapes,stretched out tentacles,and the contacts between them were loosened.When radiation dose accumulated to 24 Gy,an over-expression of Vimentin was observed in treated cells,while E-cadherin was down-regulated (P ＜ 0.01).Conclusions NPC cells present with typical morphorlogical and biomolecular changes of EMT during exposure to irradiation,indicating the potential critical roles of EMT in the malignant behavior of radioresistance in NPC.