PUMA／ASPP1双抑癌基因对胃癌 SGC-7901细胞增殖的影响

摘要

目的：探讨 p53正向细胞凋亡调控因子（p53 up-regulated modulator of apoptosis protein，PUMA）／p53促凋亡蛋白（apoptosis stimulating protein of p53，ASPP）1融合基因及单个基因对胃癌 SGC-7901细胞的杀伤作用。方法通过脂质体（lipofectamine）2000TM 将 PUMA／ASPP1融合基因及 PUMA、ASPP1单个基因分别转染胃癌SGC-7901细胞系[实验设5组，分别为胃癌细胞无转染空白对照（SGC-7901）组，空载质粒转染细胞对照（SGC-7901-pcDNA3．1）组以及重组 ASPP1质粒转染细胞实验（SGC-7901-ASPP1）组、重组 PUMA 质粒转染细胞实验（SGC-7901-PUMA）组、重组 PUMA／ASPP1质粒转染细胞实验（SGC-7901-PUMA／ASPP1）组]，再次经 G418筛选，获得稳定表达融合基因 SGC-7901-PUMA／ASPP1细胞株。通过 MTT 法及使用流式细胞仪测定各组胃癌SGC-7901系细胞的存活数（光吸收度即 A 值）及细胞周期。结果SGC-7901组与 SGC-7901-pcDNA3．1组 A 值比较差异无统计学意义（P ＞0．05）；SGC-7901-ASPP1组、SGC-7901-PUMA 组和 SGC-7901-PUMA／ASPP1组 A值明显低于 SGC-7901组与 SGC-7901-pcDNA3．1组（均 P ＜0．01）；SGC-7901-ASPP1组、SGC-7901-PUMA 组和SGC-7901-PUMA／ASPP13组中，SGC-7901-PUMA／ASPP1细胞组 A 值最低（P ＜0．01）。与 SGC-7901组、SGC-7901-pcDNA3．1组比较，SGC-7901-ASPP1、SGC-7901-PUMA、SGC-7901-PUMA／ASPP1组的 G1期明显增多，S 期明显减少，尤以 SGC-7901-PUMA／ASPP1组 S 期减少为甚（均 P ＜0．01）；SGC-7901-pcDNA3．1组与 SGC-7901组比较 G1期、S 期差异无统计学意义（P ＞0．05）。结论双抑癌基因可有效抑制胃癌 SGC-7901系细胞增殖，双抑癌基因较单个抑癌基因具有更强的抗肿瘤作用。%ABSTRACT:Objective To investigate the lethal effects of p53 up-regulated modulator of apopto-sis(PUMA),apoptosis stimulating protein of p53(ASPP1 )and PUMA-ASPP1 fusion gene on hu-man gastric cancer SGC-7901 cells.Methods PUMA gene,ASPP1 gene and PUMA-ASPP1 fusion gene were transfected into SGC-7901 cells by using the Lipofectamine 2000 TM,respectively. SGC-7901 cells were divided into five groups:no treatment(SGC-7901 group),empty plasmid transfection(SGC-7901-pcDNA3.1 group),recombinant ASPP1 plasmid transfection(SGC-7901-ASPP1 group),recombinant PUMA plasmid transfection(SGC-7901-PUMA group),and recombi-nant PUMA-ASPP1 plasmid transfection(SGC-7901-PUMA-ASPP1 group).The stable SGC-7901-PUMA-ASPP1 cells were obtained by G418 growth inhibition screen.Cell survival and cell cycle were examined by MTT and flow cytometry,respectively.Results There were no significant differences in the A value(light absorbance)and number of cells in G1/S phase between SGC-7901 group and SGC-7901-pcDNA3.1 group (P > 0.05 ).Compared with SGC-7901 group and SGC-7901-pcDNA3.1 group,the A value reduced,number of cells in G1 phase increased and num-ber of cells in S phase decreased in SGC-7901-ASPP1 group,SGC-7901-PUMA group and SGC-7901-PUMA-ASPP1 group(P <0.01).The minimum A value and number of cells in S phase were observed in SGC-7901-PUMA-ASPP1 group.Conclusion The PUMA-ASPP1 fusion gene is more effective than PUMA or ASPP1 alone for inhibiting the proliferation of gastric cancer SGC-7901 cells.