摘要目的 观察c-myc小干扰RNA(siRNA)联合氟尿嘧啶(5-Fu)抑制喉癌Hep-2细胞生长的体内外作用,探讨在喉癌治疗中将c-myc作为基因治疗靶点的价值.方法 利用RNA干扰技术将c-myc siRNA转染到喉癌Hep-2细胞中,Western blot法检测c-myc蛋白的表达,流式细胞仪检测c-myc siRNA与5-Fu单独或联合应用,对喉癌细胞周期的影响.构建裸鼠移植瘤模型,观察c-myc siRNA及联合5-Fu对肿瘤生长的影响.免疫组织化学法检测c-myc在肿瘤组织中的表达;脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(terminal-deoxynucleotidyl transferase mediated nick end labeling,TUNEL)检测肿瘤组织细胞的凋亡情况.结果 在转染c-myc siRNA后的喉痛Hep-2细胞中,c-myc 蛋白的表达水平逐渐下调;G0-G1期的细胞开始增加,同时S期的细胞逐渐减少;当转染c-myc siRNA 细胞联合使用5-Fu时,G0-G1期的细胞明显增加,为(79.3.±2.1)%,同时S期的细胞减少为(17.5±1.7)%.c-myc siRNA联合5-Fu组移植瘤生长最慢,与c-myc siRNA组和5-Fu组相比差异有统计学意义(t值分别为44.170和78.988,P＜0.05);c-myc siRNA+5-Fu组治疗的移植瘤重量明显小于对照组,也小于c-myc siRNA组和5-Fu组(P值均＜0.05).免疫组织化学结果显示,c-myc siRNA及其联合5-Fu组肿瘤组织中c-myc蛋白的表达下调,肿瘤细胞的凋亡数明显增加高于单用5-Fu组(t=-17.871,P＜0.05).结论 c-myc siRNA可特异性地下调Hep-2细胞和裸鼠组织中c-myc的表达,联合5-Fu时,可显著抑制喉癌移植瘤的生长,促进肿瘤细胞的凋亡,表明c-myc可能是喉癌基因治疗中一个重要的分子靶点.

abstractsObjective To observe the effects of small interfere RNA (siRNA) targeting the c-myc in combination with 5-fluorouracil (5-Fu) on the growth of Hep-2 cells in vitro and in vivo.Methods Hep-2 cells transfected with or without c-myc siRNA were treated with 5-Fu for 48 h.C-myc protein levels in Hep-2 cells were detected using the Western blot.The cell cycle was analyzed by flow cytometry.Hep-2 cells were subcutaneously inoculated into the back of BALB/c nude mice to establish the implanted laryngeal squamous carcinoma model.PBS,c-myc siRNA,and 5-Fu,alone or in combinations were administered i.p.The mice were sacrificed after the treatments and the tumor masses were removed to determine the tumor volume and weight.The inhibitory rate was calculated.Expression of c-myc in tumor tissue was detected by immunocytochemistry and cell apoptosis was analyzed by terminal transferase dUTP nick end labeling (TUNEL).Results The protein levels of c-myc decreased after transfected with c-myc siRNA.C-myc siRNA-transfected cells showed an increase in the percentage of cells in the G0-G1 phase and a decrease in the percentage of cells in the S phase.When combined with 5-Fu,the results were improved.The tumor growth was faster in the control group and was significantly slower in the c-myc siRNA plus 5-Fu group than that in the c-myc siRNA group or 5-Fu group ( P ＜0.05 ) .The tumor weight in the c-myc siRNA plus 5-Fu group was significantly smaller than that in the c-myc siRNA or 5-Fu group ( P ＜ 0.05 ).Immunohistochemistry showed that c-myc siRNA inhibited the expression of c-myc in tumor tissues in the c-myc siRNA group and c-myc siRNA plus 5-Fu group (P ＜0.05).The number of apoptotic cells in the c-myc siRNA plus 5-Fu group was higher than those in the c-myc siRNA groups (P ＜0.05).Conclusions C-myc siRNA inhibits the expression of c-myc in Hep-2 cells and in the tumor tissues of nude mice.C-myc siRNA combined with 5-Fu inhibits the growth of implanted laryngeal squamous carcinoma and promotes cell apoptosis.C-myc could become a novel target for the treatment of laryngeal squamous carcinoma.