摘要
目的观察反义DNA甲基转移酶3b(DNMT3b)基因真核表达载体转染对人胆管癌QBC939细胞中DNMT3b基因表达的影响。方法构建反义DNMT3b基因真核表达载体,通过脂质体转染到人胆管癌细胞株QBC939中,G418筛选得到稳定转染细胞株,PCR鉴定外源性neoR基因在转染细胞中的表达以确定转染是否成功。半定量RTPCR观察转染前后DNMT3b基因mRNA水平的变化,流式细胞术检测转染前后DNMT3b蛋白的变化。结果反义DNMT3b基因真核表达载体转染能使人胆管癌QBC939细胞中DNMT3b基因的mRNA水平从0.956±0.053降低至0.209±0.023,差异有统计学意义(P<0.01);流式细胞术检测结果显示,DNMT3b蛋白水平从(75.38±3.22)%降低到(29.87±3.46)%,差异有统计学意义(P<0.01)。结论反义DNMT3b基因转染能降低人胆管癌QBC939细胞中DNMT3b基因的表达水平,为研究DNMT3b基因功能及其在胆管癌细胞中的作用提供了方法和实验依据。
Objective To study the effect of transfection with antisense DNMT3b gene eukaryotic expression vector on the expression of DNMT3b gene in human cholangiocarcinomacellline QBC-939. Methods The constructed antisense DNMT3b gene eukaryotic expression vector was transfeeted into the human cholangioeareinoma cell line QBC-939 by using lipofeetaminc transfeetion reagents, and positive cell clones were obtained by using G418 selection after transfection. Whether the constructed recombinant vector was transfeeted into QBC-939 cells successfully was confirmed hy amplifying the exogenous neo^k gene with PCR method. The expression of DNMT3b gene mRNA and protein were detected by semi-quantitative RT-PCR and FCM methods respectively. Results Following the transfection of antisense DNMT3b gene eukaryotic expression vector, the mRNA level of DNMT3b gene in QBC-939 cells of human cholangiocarcinoma decreased from 0. 956±0. 053 to 0. 209±0. 023, and the protein level of DNMT3h gene also decreased from （75.38±3.22）% to （29.87±3.46）%. There were very significant differences on the expression levels of DNMT3b gene between non-tranfections group and the antisense DNMT3b gene eukaryotic expression vector transfection group （P〈0.01）. Conclusion Transfeetion with antisense DNMT3b gene eukaryolic expression vector significantly reduces the expression level of DNMT3b gene in human eholangioeareinoma cell line QBC-939, and this study may provide a valid tool and method to investigate the function of DNMT3b gene and its role in cholangiocarcinoma.
出处
《中国普外基础与临床杂志》
CAS
2006年第1期56-60,共5页
Chinese Journal of Bases and Clinics In General Surgery
基金
国家高技术研究发展计划(863计划
编号:2002AA214061)
