兰州大学机构库 >学院待认领
siRNA沉默人胃癌细胞COX-2基因对Notch信号通路影响的研究
Alternative TitleThe effect of silencing COX-2 genes on Notch signaling pathway in human gastric carcinoma cells
王璟
Thesis Advisor刘宏斌
2014-05-23
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword胃肿瘤 环氧合酶-2 RNA干扰 Notch信号通路
Abstract目的:1.研究siRNA沉默COX-2基因表达对人胃癌细胞增殖和凋亡能力的影响。2.研究siRNA沉默COX-2基因表达对人胃癌细胞Notch信号通路的影响及相关机制。 方法:siRNA干扰技术沉默胃癌MKN45细胞 COX-2基因,将实验分为三组,转染siRNA-COX-2的胃癌细胞为实验组,转染siRNA Control为阴性对照组,以未转染的MKN45细胞为空白组。应用荧光定量-PCR检测靶向沉默COX-2基因的表达和沉默效率,以及Notch信号通路相关基因的表达水平;蛋白免疫印记法检测COX-2,N1ICD,N2ICD和Hes1蛋白表达水平;细胞计数法检测各组细胞增殖,流式细胞术检测分析各组细胞凋亡。 结果:转染siRNA沉默COX-2基因可明显下调COX-2的表达量,实验组与阴性对照组比较差异有统计学意义(P<0.05),表达抑制率为(81.81±3.82)%;Notch信号通路下游基因Hes1 mRNA表达水平也下调,差异有统计学意义(P<0.05),表达抑制率为(74.01±4.35)%;Notch1和Notch2 mRNA表达水平无明显变化,与阴性对照组比较,差异无统计学意义(P>0.05);沉默COX-2基因后COX-2,N1ICD,N2ICD和Hes-1蛋白表达明显受到抑制,实验组与阴性对照组比较,差异有统计学意义(P<0.05)。细胞计数法检测在转染后第4天细胞增值能力明显下降(P<0.05);沉默COX-2基因可提高细胞凋亡率,转染48小时后实验组与阴性对照组比较,差异有统计学意义(P<0.05)。 结论:靶向沉默COX-2基因抑制人胃癌细胞增殖和促进细胞凋亡可能是通过抑制Notch信号通路实现。
Other AbstractObjective: 1. To investigate the effect of silencing COX-2 gene on proliferation and apoptosis. 2. To investigate the underlying mechanisms with Notch signaling pathway in human gastric carcinoma cell line by target RNA interference. Methods: The siRNA of COX-2 was transfected into gastric cancer cell line MKN45 by Lipofectamine 2000. MKN45 cells transfected with blank vector were served as negative control group. MKN45 cells non-transfected served as blank group. Application of qRT-PCR to detect the COX-2 gene expression and silencing efficiency. The mRNA expression of Notch1, Notch2 and Hes-1 were determined by qRT-PCR. The protein expressions of COX-2, N1ICD, N2ICD and Hes-1 were determined by Western blotting. Proliferation of MKN45 was detected after transfection by cell counting method. Cell apoptosis was detected by flow cytometry before and after transfection. Results: The mRNA expression of COX-2 and Hes-1(P<0.05) but not Notch1 and Notch2(P>0.05) were significantly decreased after transfection. Inhibition of COX-2 gene expression rate was (81.81 ± 3.82)% and inhibition of Hes1 gene expression rate was (74.01 ± 4.35)%. The protein expressions of COX-2, N1ICD, N2ICD and Hes-1 were obviously inhibited(P<0.05) and the difference was statistically significant. Compared with experiment group and negative group, it can Inhibit cell proliferation and induce apoptosis after COX-2 silencing.The difference was statistically significant (P< 0.05). Conclusion: RNA silence targeting COX-2 can inhibit cell proliferation and induce apoptosis of gastric cell line probably by inhibiting Notch signaling pathway.
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/223929
Collection学院待认领
Affiliation临床医学院
Recommended Citation
GB/T 7714
王璟. siRNA沉默人胃癌细胞COX-2基因对Notch信号通路影响的研究[D]. 兰州. 兰州大学,2014.
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