中华放射肿瘤学杂志
中華放射腫瘤學雜誌
중화방사종류학잡지
CHINESE JOURNAL OF RADIATION ONCOLOGY
2014年
1期
73-77
,共5页
毛细血管扩张共济失调突变基因%RNA干扰%细胞系,肺腺癌%放射敏感性
毛細血管擴張共濟失調突變基因%RNA榦擾%細胞繫,肺腺癌%放射敏感性
모세혈관확장공제실조돌변기인%RNA간우%세포계,폐선암%방사민감성
Ataxia-telangiectasia mutated gene%RNA interference%Cell line,lung adenocarcinoma%Radiosensitivity
目的 探讨质粒介导RNA干扰抑制ATM基因表达对人肺腺癌A549细胞放射敏感性影响.方法 构建ATM基因小分子干扰RNA (siRNA)真核表达质粒pSilencer2.1-ATM并转染A549细胞(阳性组),转染pSilencer2.1-nonspecific质粒为阴性组,未转染为对照组.RT-PCR和蛋白印迹法分别检测ATM基因mRNA及蛋白表达,细胞克隆形成实验观察细胞放射敏感性变化,流式细胞仪检测细胞周期和细胞凋亡.结果 成功构建了ATM基因siRNA真核表达质粒.RT-PCR和蛋白印迹法证实阳性组细胞ATM基因表达下调,阳性组、阴性组的放射增敏比(D0值比)分别为1.50、1.01,流式细胞仪检测显示阳性组G1、G2 +M期细胞比例减少[51.27％∶ 61.85％ (P =0.012)、6.34％∶ 10.91％(P=0.008)],细胞凋亡率则高于对照组、阴性组[49.31％∶ 13.58％ (P=0.000)、49.31％∶ 13.17％(P=0.000)].结论 ATM基因沉默可增加A549细胞的放射敏感性,其机制可能与细胞周期变化及凋亡有关.
目的 探討質粒介導RNA榦擾抑製ATM基因錶達對人肺腺癌A549細胞放射敏感性影響.方法 構建ATM基因小分子榦擾RNA (siRNA)真覈錶達質粒pSilencer2.1-ATM併轉染A549細胞(暘性組),轉染pSilencer2.1-nonspecific質粒為陰性組,未轉染為對照組.RT-PCR和蛋白印跡法分彆檢測ATM基因mRNA及蛋白錶達,細胞剋隆形成實驗觀察細胞放射敏感性變化,流式細胞儀檢測細胞週期和細胞凋亡.結果 成功構建瞭ATM基因siRNA真覈錶達質粒.RT-PCR和蛋白印跡法證實暘性組細胞ATM基因錶達下調,暘性組、陰性組的放射增敏比(D0值比)分彆為1.50、1.01,流式細胞儀檢測顯示暘性組G1、G2 +M期細胞比例減少[51.27％∶ 61.85％ (P =0.012)、6.34％∶ 10.91％(P=0.008)],細胞凋亡率則高于對照組、陰性組[49.31％∶ 13.58％ (P=0.000)、49.31％∶ 13.17％(P=0.000)].結論 ATM基因沉默可增加A549細胞的放射敏感性,其機製可能與細胞週期變化及凋亡有關.
목적 탐토질립개도RNA간우억제ATM기인표체대인폐선암A549세포방사민감성영향.방법 구건ATM기인소분자간우RNA (siRNA)진핵표체질립pSilencer2.1-ATM병전염A549세포(양성조),전염pSilencer2.1-nonspecific질립위음성조,미전염위대조조.RT-PCR화단백인적법분별검측ATM기인mRNA급단백표체,세포극륭형성실험관찰세포방사민감성변화,류식세포의검측세포주기화세포조망.결과 성공구건료ATM기인siRNA진핵표체질립.RT-PCR화단백인적법증실양성조세포ATM기인표체하조,양성조、음성조적방사증민비(D0치비)분별위1.50、1.01,류식세포의검측현시양성조G1、G2 +M기세포비례감소[51.27％∶ 61.85％ (P =0.012)、6.34％∶ 10.91％(P=0.008)],세포조망솔칙고우대조조、음성조[49.31％∶ 13.58％ (P=0.000)、49.31％∶ 13.17％(P=0.000)].결론 ATM기인침묵가증가A549세포적방사민감성,기궤제가능여세포주기변화급조망유관.
Objective To investigate the effect of silencing of ataxia-telangiectasia mutated (ATM) expression by plasmid-mediated RNA interference on the radiosensitivity of human lung adenocarcinoma A549 cells.Methods Eukaryotic expression plasmid containing ATM small interfering RNA (siRNA) (pSilencer2.1-ATM),as well as pSilencer2.1-nonspecific,was constructed.Lung adenocarcinoma A549 cells were divided into positive group,negative group,and control group to be transfected with pSilencer2.1-ATM,pSilencer2.1-nonspecific,and no plasmid,respectively.The mRNA and protein expression of ATM was measured by RT-PCR and Western blot,respectively.The change in cell radiosensitivity was observed by colony-forming assay.Cell cycle and cell apoptosis were analyzed by flow cytometry.Results The eukaryotic expression plasmid containing ATM siRNA was successfully constructed.The RT-PCR and Western blot demonstrated that the expression of ATM was down-regulated in the positive group.The sensitization enhancement ratios (D0 ratios) for the positive group and negative group were 1.50 and 1.01,respectively.The flow cytometry revealed that the proportions of A549 cells in G1 and G2/M phases were significantly lower in the positive group than in the control group (51.27％ vs 61.85％,P =0.012;6.34％ vs 10.91％,P =0.008) and that the apoptosis rate was significantly higher in the positive group than in the control group and negative group (49.31％ vs 13.58％,P=0.000;49.31％ vs 13.17％,P=0.000).Conclusions Silencing of ATM expression may increase the radiosensitivity of human lung adenocarcinoma A549 cells,probably by affecting the cell cycle and promoting cell apoptosis.
