利用RNA干扰技术探讨ATM基因沉默对大肠癌HT29细胞的放射敏感性.doc

PAGE PAGE 39 利用RNA干扰技术探讨ATM基因沉默对大肠癌HT29细胞的放射敏感性 摘要 目的通过大肠癌体外细胞模型，利用RNA干扰技术，探讨ATM基因沉默是否对大肠癌HT29细胞的放射敏感性有影响。 方法设计并构建ATM基因小分子干扰RNA(Small interfering RNA, siRNA)真核表达质粒pSilencer2.1-ATM，并转染人大肠癌HT29细胞株作为阳性组；ATM基因RNAi寡聚脱氧核糖核酸序列随机重新排列后构建非特异性siRNA，并转染pSilencer2.1-nonspecific质粒作为阴性组，未转染为对照组。各组分别采用逆转录-聚合酶链反应(Reverse Transcription-Polymerase Chain Reaction, RT-PCR)检测各组ATM基因的mRNA含量，蛋白质免疫印迹法(Western blot, WB)检测每组ATM基因mRNA表达的蛋白质含量，各组细胞经8 Gy X线照射后，分成两部分，分别继续培养24 h、48 h，然后采用流式细胞仪检测不同时间点(24 h、48 h)的阳性组、阴性组以及对照组的细胞周期分布和细胞凋亡情况。 结果 ATM基因siRNA真核重组表达质粒构建后成功转染大肠癌HT29细胞。RT-PCR检测证实阳性组ATM基因的mRNA含量明显下降；Western blot结果证实阳性组细胞ATM基因所表达蛋白量明显减少。经X线照射后24h，阳性组G1+G0期、G2+M期细胞比例较对照组均减少(t= -11.59, P＜0.001; t= -4.30, P＜0.001)，但阴性组与对照组相比无明显差别(t=0.02, P=0.98; t= -0.03, P=0.97)；照射48h后，G1+G0期、G2+M期细胞比例同样较对照组减少(t= -9.91, P＜0.001; t= -4.94, P＜0.001)，阴性组与对照组同样无明显差别(t=0.08, P=0.94; t=0.09, P=0.93)；照射后24、48 h阳性组细胞凋亡率均高于对照组(t=21.15, P＜0.001; t=32.94, P＜0.001)，阴性组与对照组相比无明显差别(t=1.56, P=0.12; t=1.86, P=0.06)。 结论 1. 通过构建ATM基因siRNA的真核重组表达质粒，并转染人大肠癌HT29细胞，有效抑制了ATM基因的转录以及表达。 2. ATM基因被沉默后，增强了大肠癌HT29细胞的放射敏感性，其机制可能与抑制DNA损伤修复、使G1+G0期和G2+M期细胞周期检测点失活、增加细胞凋亡率有关。 关键词 ATM基因；RNA干扰；大肠癌；放射敏感性 ABSTRACT OBJECTIVE Using RNA interference technique to investigate the effect of ATM gene silencing on the radiosensitivity of colorectal cancer HT29 cells in vitro. METHODS Small interfering RNA(siRNA) of ATM gene, eukaryotic expression plasmid called pSilencer2.1-ATM, was designed and constructioned to transfect into human colorectal cancer HT29 cells as positive group. RNAi oligonucleotide sequence of ATM gene was rearranged randomly to construct pSilencer2.1-nonspecific plasmid as negative group. Control group was not transfected. The transcription of ATM gene mRNA in each group was determined with Reverse Transcription-Polymerase Chain Reaction(RT-PCR). Protein expression of ATM gene mRNA in each group was detected by Western blot(WB). After 8 Gy X-ray irradiation, each group was divided into two halves. One was cultured continuously for