【作者】 徐寒子；

【导师】 王仁生；

【作者基本信息】 广西医科大学 ， 肿瘤放射治疗， 2009， 硕士

【摘要】 第一部分:西妥昔单抗诱导人鼻咽癌细胞株CNE凋亡实验研究目的:观察西妥昔单抗诱导人鼻咽癌细胞株CNE凋亡的效果,并初步探讨其可能机制。方法:MTT法测定西妥昔单抗对CNE细胞株20～30%抑制的工作浓度;以该浓度药物作用CNE细胞,透射电镜、Hochest33258荧光染色法观察其凋亡形态学变化,流式细胞术检测凋亡率;western blot检测Bax、Bcl-2蛋白的表达。结果:西妥昔单抗对CNE细胞株的工作浓度(IC20～30)为10ug/ml;CNE细胞经西妥昔单抗处理后,电镜、荧光显微镜下均可见典型的凋亡形态学及超微结构改变;流式细胞术显示24、48h凋亡率分别为14.30±0.78%、15.30±0.95%,显著高于对照组(P<0.05);western blot检测显示Bax蛋白表达上调、Bcl-2蛋白表达下调、Bax/Bcl-2比值增加。结论:西妥昔单抗对人鼻咽癌细胞株CNE有显著的凋亡诱导作用,这可能与其上调Bax/Bcl-2比值促进凋亡有关。第二部分:西妥昔单抗联合热疗诱导人鼻咽癌细胞株CNE凋亡实验研究目的:观察西妥昔单抗联合热疗诱导人鼻咽癌细胞株CNE凋亡的效果,并初步探讨其可能的机制。方法:试验分单靶组、单热组及热靶组,Hochest33258荧光染色法观察CNE细胞株各处理组细胞凋亡形态学变化;流式细胞术检测凋亡率;western blot检测CNE细胞株不同处理后Bax、Bcl-2、EGFR以及热疗后不同时间EGFR蛋白的表达。结果:荧光染色法观察到CNE细胞发生典型的凋亡形态学改变,以热靶组更为显著;流式凋亡检测显示热靶组凋亡率显著高于单靶组、单热组及对照组(P<0.05);western blot检测显示各处理组较之对照组都有上调Bax/Bcl-2比值、下调EGFR蛋白表达作用,以热靶组最为显著(P<0.05);热疗后随时间的延长,EGFR蛋白表达逐渐下调。结论:西妥昔单抗联合低温热疗能显著增加CNE细胞的凋亡率,这可能与EGFR的下调与受抑,进而调变Bax/Bcl-2比值进一步促进凋亡有关。第三部分:西妥昔单抗联合放疗及热疗诱导人鼻咽癌细胞株CNE凋亡实验研究目的:观察西妥昔单抗联合放疗及低温热疗体外诱导人鼻咽癌细胞株CNE凋亡的效果,并初步探讨其可能的机制。方法:试验分对照组、单放组、热放组、靶放组及热靶放组,Hochest 33258荧光染色法观察CNE细胞株细胞凋亡形态学变化;流式细胞术检测凋亡率; Western blot检测各组Bax、Bcl-2蛋白的表达。结果:荧光染色法观察到CNE细胞发生典型的凋亡形态学改变,以靶放热组更为显著;流式凋亡检测显示各照射剂量下靶放热组凋亡率显著高于对照组及相应单放组、热放组、靶放组(P<0.05);western blot检测显示各处理组较之对照组都有上调Bax/Bcl-2比值,以靶放热组最为显著(P<0.05)。结论:西妥昔单抗联合放疗和低温热疗显著增加CNE细胞的凋亡率,这可能与其上调Bax/Bcl-2比值进一步促进凋亡有关。更多还原

【Abstract】 Part One: The experimental study on apoptosis of human nasopharyngeal carcinoma CNE cell line induced by cetuximab in vitro Objective: To investigate the apoptosis mechanism of CNE cell line induced by cetuximab in vitro. Methods: The working concentration of cetuximab was defined as its IC(20～30) at 48 hours action against CNE which was determined by MTT assay. Cells apoptosis was assayed by transmission electron microscopy(TEM), fluorescent Hocehst 33258 staining and flow cytometric analysis(FCM). The expression of Bcl- 2、Bax proteins were determined by Western Blot. Results: A working concentration of Cetuximab at 10μg/ml was obtained and subsequently used in the following experiments. Characteristical changes of apoptosis in CNE cells were observed and the apoptosis rate of 24, 48h was 14.30±0.78%, 15.30±0.95% respectively(P<0.05 compared with control group). In addition, Western Blot analysis indicated the level of expression of the proteins Bax significantly increased while Bcl-2 decreased by the treatment. Conclusion: Cetuximab could significantly induce the apoptosis of CNE cells, possibly through the Bax up-regulation and Bcl-2 down-regulation. Part Two : The experimental study on apoptosis of human nasopharyngeal carcinoma CNE cell line induced by cetuximab combined with hyperthermia in vitroObjective: To investigate the apoptosis mechanism of CNE cell line induced by cetuximab combined with hyperthermia in vitro. Methods: Apoptosis was assayed by fluorescent Hochest 33258 staining and flow cytometry(FCM) in three experimental groups: cetuximab(10ug/ml) or hyperthermia(43℃, 30min) alone, cetuximab combined with hyperthermia. The expression of Bax, Bcl-2, EGFR proteins were determined by Western Blot. Results: Characteristical changes of apoptosis in CNE cells were observed in all experimental groups, especially in cetuximab combined with hyperthermia group. FCM also showed more prominent apoptosis induced by cetuximab combined with hyperthermia group compared to cetuximab or hyperthermia alone groups. Western Blot analysis indicated that the ratio of Bax/Bcl-2 significantly increased while the expression of the protein EGFR decreased by the treatment,the level of expression of the protein EGFR decreased gradually after the treatment of hyperthermia. Conclusion: Cetuximab combined with hyperthermia synergistically increases the apoptosis in CNE cells, possibly by further down-regulating expression of EGFR to up-regulate Bax/Bcl-2 ratio.Part Three : The experimental study on apoptosis of human nasopharyngeal carcinoma CNE cell line induced by cetuximab in conjunction with radiotherapy and hyperthermia in vitroObjective: To investigate the apoptosis mechanism of CNE cell line induced by Cetuximab in conjunction with radiotherapy and hyperthermia in vitro. Methods:Apoptosis was assayed by fluorescent Hocehst 33258 staining and flow cytometry (FCM) in five experimental groups: control with no treatment (C), radiotherapy alone(2、4、8、12Gy)(R), Cetuximab with radiotherapy(TR), Cetuximab with hyperthermia(43℃, 30min)(TH), and Cetuximab with radiotherapy plus hyperthermia(TRH). The expression of Bcl-2, Bax proteins were determined by Western Blot. Results: Characteristical changes of apoptosis in cells treated with cetuximab were observed in all the experimental groups, especially in TRH. FCM also showed more prominent apoptosis induced by TRH group compared to the control, R, TR and RH groups. In addition, Western Blot analysis indicated that the ratio of the proteins Bax/Bcl-2 by the treatment. Conclusion: Cetuximab in conjunction with radiotherapy and hyperthermia synergistically increases the apoptosis in CNE cells, possibly through further up-regulation of the ratio Bax/Bcl-2.更多还原