ATM基因沉默对大肠癌细胞放射敏感性影响的实验研究

发布时间：2018-04-22 19:59

本文选题：ATM基因 + RNA干扰　；参考：《延边大学》2016年硕士论文

【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8Gy 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+Go期、G2+M期细胞比例较对照组均减少(t=-11.59,P0.001；t=-4.30,P0.001),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+Go期、G2+M期细胞比例同样较对照组减少(t=-9.91,P0.001；t=-4.94,P0.001),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,P0.001；t=32.94,P0.001),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。
[Abstract]:Objective to investigate the effect of ATM gene silencing on radiosensitivity of colorectal cancer HT29 cells by RNA interference in vitro. Methods the eukaryotic expression plasmid pSilencer2.1-ATM was designed and constructed for ATM gene small molecule interference RNA(Small interfering, and then transfected into human colorectal cancer HT29 cell line as a random rearrangement of RNAi oligodeoxyribonucleic acid sequence to construct nonspecific siRNAs. PSilencer2.1-nonspecific plasmid was transfected as negative group. No transfection was used as control group. Reverse Transcription-Polymerase Chain reaction (RT-PCR) was used to detect the mRNA content of ATM gene in each group, and the protein content of ATM gene mRNA in each group was detected by Western blotting. The cells in each group were irradiated by 8Gy X ray. The cell cycle distribution and apoptosis were detected by flow cytometry in the positive group, the negative group and the control group. Results after the construction of the eukaryotic expression plasmid of ATM gene siRNA, the mRNA content of ATM gene in the positive group was significantly decreased. The results showed that the expression of ATM protein in the positive group was significantly decreased. 24 hours after X-ray irradiation, the percentage of G 1 go phase G 2 M cells in the positive group was lower than that in the control group, but there was no significant difference between the negative group and the control group, but there was no significant difference between the negative group and the control group. 48 h after irradiation, the proportion of G 2 M phase cells in G 1 go phase in the positive group was also lower than that in the control group, and there was no significant difference between the negative group and the control group, and the proportion of the G 2 phase cells in the G 1 phase of G 1 go phase was also lower than that in the control group, and the percentage of the cells in the G 2 phase in the G 1 go phase was also lower than that in the control group, and there was no significant difference between the negative group and the control group. There was no significant difference between the control group and the control group, and there was no significant difference between the control group and the control group, and there was no significant difference between the negative group and the control group, and the apoptosis rate of the positive group was higher than that of the control group at 24 h after 48 h irradiation. The rate of apoptosis in the negative group was higher than that in the control group. There was no significant difference between the negative group and the control group, and there was no significant difference between the negative group and the control group. Conclusion 1. By constructing the eukaryotic expression plasmid of ATM gene siRNA and transfection into human colorectal cancer HT29 cells, the transcription of ATM gene was effectively inhibited and the expression of .2. siRNA gene was silenced, which enhanced the radiosensitivity of HT29 cells. The mechanism may be related to the inhibition of DNA damage and repair, the inactivation of cell cycle detection points in G1 G0 phase and G 2 M phase, and the increase of apoptosis rate.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.34

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