重组腺病毒介导PUMA基因对胰腺癌细胞的放射增敏作用

Radiosensitization effect of recombinant adenoviral-mediated PUMA gene on pancreatic carcinoma cells

目的探讨重组腺病毒（Ad）介导PUMA基因联合γ线对人胰腺癌的作用。方法以不同感染复数（MOI）的Ad-PUMA（10、50、100）转染胰腺癌PANC-1细胞，RT-PCR和Westernblot检测转染前后细胞内PUMA表达。PANC-1细胞分为4组：对照组、转染组、照射组、转染联合照射组。MTT比色法和流式细胞术检测各组细胞存活率和凋亡率。人胰腺癌裸鼠皮下移植瘤模型分为4组：对照组、转染组、照射组、转染联合照射组，在不同时间测量肿瘤生长速率和凋亡指数。结果PUMA表达随病毒MOI增加而进行性增加（MOI=10、mRNA=0．46±0．02、蛋白：0．75±0．09；MOI=50、mRNA=1．12±0．09、蛋白=1．01±0．18；MOI=100、mRNA=1．50±0．08、蛋白=1．80±0．15；P〈0．05），细胞增殖随病毒MOI增加逐渐受抑制（r=-0．98655），经7线照射后增殖抑制更加明显（r=-0．97126，P〈0．05），而凋亡率上升（照射前=45．4％±5．26％，照射后=73．2％±6．62％，P〈0．05）。Ad—PUMA转染和γ线联合照射后7～35d，裸鼠肿瘤生长速率明显低于单纯照射组、单纯转染组和对照组[第35天肿瘤体积分别为（19．82±6．45）、（39．5±9．23）、（33．6±10．3）、（52．0±11．43）mm^3，P〈0．05]，凋亡指数升高（A1分别为0．43±0．05、0．29±0．10、0．24±0．05、0．00±0．00，P〈0．05）。结论PUMA基因转染联合γ线照射可增强对胰腺癌细胞杀灭作用，联合治疗明显优于单纯照射和单纯基因治疗。

Objective To study the effect of PUMA gene mediated by recombinant adenovirus vector combined with radiation on the pancreatic carcinoma. Methods The PANC-1 cells were infected with AdPUMA （MOI = 10, 50 and 100, respectively） for 48 h. The expression of PUMA mRNA and protein was detected by RT-PCR and Western blot, respectively. PANC-1 ceils were divided into 4 groups： control group, transfection group, irradiation group and combined treatment group. The cell growth inhibition rate and apoptotic rate of PANC-1 cells were assessed by MTT assay and flow cytometry. Human pancreatic carcinomas were transplanted subcutaneously in nude mice, which were randomized into 4 groups： control group, transfection group, irradiation group and combined treatment group. Tumor growth rate and apoptotic index at different time points were recorded in 35 days. Results The expression of PUMA mRNA and protein was increased with the increase of MOI of Ad-PUMA, which was does-dependant （MOI = 10, mRNA = 0.46 ± 0.02, protein = 0.75 ± 0.09; MOI=50, mRNA=1.12±0.09, protein =1.01±0.18; MOI=100, mRNA=1.50±0.08, protein= 1.80 ± 0.15 ; P 〈 0.05）. The proliferation of PANC-1 cells was suppressed significantly when transfected by Ad- PUMA in a dose-dependent manner（ r = - 0.986 55）, which was more significant combined with radiation （ r = -0.971 26,P 〈 0.05）. Meanwhile, the apoptotic rate was increased in the same manner [for pre- and postirradiation,which was （45.4 ± 5.26） % and （73.2 ± 6.62） %, respectively, P 〈 0.05 ]. From 7 to 35 d after PUMA gene transfection and radiotherapy, the tumor growth was significantly slower than those of irradiation group, transfection group and control group [35 d after therapy, the volume of tumor was （19.82 ± 6.45）mm^3 , （39.5 ± 9.23）mm^3 , （33.6 ± 10.3）mm^3 and （52.0 ± 11.43）mm^3 , respectively, P 〈 0.051. And the apoptotic index was increased in the same manner （AI = 0.43 ± 0.05, 0.29 ± 0.10, 0.24 ± 0.05 and 0.00 ± 0.00, respectively, P 〈 0.05）. Conclusions Recombinant adenoviral-mediated PUMA gene combined with irradiation could increase the cell-killing effect on pancreatic carcinoma. It is better than that of either one kind of therapy.