CAJ | 학술논문

目的探讨自噬现象在照射致人鼻咽低分化鳞癌(CNE-2)细胞死亡过程中的作用.方法采用Western blot技术检测CNE-2细胞经射线照射后自噬标志物LC3、P62的变化,透射电镜检测自噬小体；流式细胞术检测CNE-2细胞凋亡率；MTT法检测CNE-2细胞照射后不同时间的存活率；细胞克隆形成实验检测CNE-2细胞的存活能力及放射敏感性.结果透射电镜检测示自噬抑制剂磷酸氯喹(CDP)抑制照射所致自噬体的形成,自噬诱导剂雷帕霉素(RAPA)增加照射致自噬体数量(F=105.15,P＜0.05)；CDP抑制射线引起的LC3-Ⅰ向LC3-Ⅱ的转化,而RAPA促进LC3-I向LC3-Ⅱ的转化(F =231.68,P＜0.05)；CDP使P62表达上调,RAPA使P62表达下调(F=117.52,P ＜0.05)；CDP+照射组和RAPA+照射组的CNE-2细胞凋亡率明显高于单纯照射组(F=143.72,P＜0.05)；CDP、RAPA降低了照射后CNE-2细胞的存活率(F=25.88,P＜0.05)；二次线性模型拟合剂量存活曲线示自噬抑制剂CDP、RAPA可增加CNE-2细胞株的放射敏感性(F=167.32,P＜0.05).结论照射联合自噬抑制剂CDP显著增加了CNE-2细胞株对射线的敏感性,提示自噬抑制剂或可用于鼻咽癌的辅助治疗.
목적 탐토자서현상재조사치인비인저분화린암(CNE-2)세포사망과정중적작용.방법 채용Western blot기술검측CNE-2세포경사선조사후자서표지물LC3、P62적변화,투사전경검측자서소체；류식세포술검측CNE-2세포조망솔；MTT법검측CNE-2세포조사후불동시간적존활솔；세포극륭형성실험검측CNE-2세포적존활능력급방사민감성.결과 투사전경검측시자서억제제린산록규(CDP)억제조사소치자서체적형성,자서유도제뢰파매소(RAPA)증가조사치자서체수량(F=105.15,P＜0.05)；CDP억제사선인기적LC3-Ⅰ향LC3-Ⅱ적전화,이RAPA촉진LC3-I향LC3-Ⅱ적전화(F =231.68,P＜0.05)；CDP사P62표체상조,RAPA사P62표체하조(F=117.52,P ＜0.05)；CDP+조사조화RAPA+조사조적CNE-2세포조망솔명현고우단순조사조(F=143.72,P＜0.05)；CDP、RAPA강저료조사후CNE-2세포적존활솔(F=25.88,P＜0.05)；이차선성모형의합제량존활곡선시자서억제제CDP、RAPA가증가CNE-2세포주적방사민감성(F=167.32,P＜0.05).결론 조사연합자서억제제CDP현저증가료CNE-2세포주대사선적민감성,제시자서억제제혹가용우비인암적보조치료.
Objective To investigate the role of autophagy in radiation-induced death response of human nasopharyngeal carcinoma cells.Methods MTT method was used to detect cell viability of CNE-2 cells in different time after irradiation.Clonogenic survival assay was used to evaluate the effect of autophagy inhibitor (chloroquine phosphate) and autophagy inductor (rapamycin) on radiosensitivity of nasopharyngeal carcinoma cells.Cell apoptosis was assessed by flow cytometry.The expressions of LC3 and P62 were measured with Western blot.Cell ultrastructural analysis was performed under an electron microscope.Results Irradiation with 10 Gy induced a massive accumulation of autophagosomes accompanied with up-regulation of LC3-Ⅱ expression in CNE-2 cells.Compared with radiation alone,chloroquine phosphate (CDP) enhanced radiosensitivity significantly by decreasing cell viability (F =25.88,P ＜ 0.05),autophagic ratio (F =105.15,P ＜ 0.05),and LC3-Ⅱ protein level(F =231.68,P ＜0.05),while up-regulating the expression of P62 (F =117.52,P ＜ 0.05).Inhibition of autophagy increased radiation-induced apoptosis (F =143.72,P ＜ 0.05).Rapamycin (RAPA) also significantly decreased cell viability,but increased autophagic ratio and LC3-Ⅱ protein level while down-regulated the expression of P62.Induction of autophagy increased radiation-induced apoptosis(F =167.32,P ＜ 0.05).Conclusions Blockage of autophagy with CDP could enhance radiosensitivity in human nasopharyngeal carcinoma cells,suggesting that inhibition of autophagy could be used as an adjuvant treatment to nasopharyngeal carcinoma.