反义MBD1基因真核表达载体转染对人胆管癌细胞MBD1基因表达的影响

Effect of transfection of antisense MBD1 gene eukaryotic expression vector on the expression of MBD1 gene in human cholangiocarcinoma cell line

目的探讨反义MBD 1基因真核表达载体转染对人胆管癌QBC-9 3 9细胞中MBD 1基因表达的影响,为进一步研究该基因的作用提供依据。方法构建反义MBD 1基因真核表达载体,通过脂质体转染入人胆管癌细胞株QBC-9 3 9中,以G 4 1 8进行筛选得到稳定转染细胞株,用PCR鉴定外源性neoR基因在转染细胞中的表达以确定转染成功。半定量RT-PCR观察转染前后MBD 1基因mRNA水平的变化,流式细胞术检测转染前后MBD 1蛋白的变化。结果人胆管癌QBC-9 3 9细胞中MBD 1基因的mRNA水平从0.9 1 2±0.0 2 2降低至0.2 1 5±0.0 1 7,转染前后的差异有显著性(P<0.01);MBD 1蛋白水平从(8 0.1 9±5.0 5)%降低到(3 5.1 1±4.0 5)%,转染前后的差异有显著性(P<0.01)。结论反义MBD 1基因转染能降低人胆管癌QBC-9 3 9细胞中MBD 1基因的表达水平,提示MBD 1基因在胆管癌的发生发展中具有重要作用,反义MBD 1基因转染有可能成为胆管癌治疗的一种新方法。

Objective To study the effect of transfection of antisense MBD1 gene eukarvntie expression vector on the expression of MBD1 gene in human eholangioeareinoma cell line QBC-939. Methods The constructed antisense MBD1 gene eukarvotie expression vector was transfeeted into the human eholangioeareinoma cell line QBC-939 using lipofeetamine transfeetinn reagents, and positive cell clones were obtained using G418 selection after transfeetion. The constnleted recombinant vector was transfeeted into QBC-939 cells successfully and was confirmed by amplifying the exogenous neo^it gene with PCR method. The expression level of MBDI gene mRNA and protein was deteeted by RT-PCR and FCM methods respectively. Results Following the transfeetion, the MBDI gene mRNA level in human eholangioeareinoma cell line QBC-939 decreased from 0. 912 ±0. 022 to 0. 215 ± 0. 017, and the MBDI gene protein level also decreased from （ 80. 19 ± 5. 05 ）% to （ 35. 11±4. 05 ）% . There were very significant differences on the expression both at the transcription and post-transcription levels of MBDI gene between non-tranfeetion group and the antisense MBD1 gene eukarvotie expression vector transfeetion group （ P 〈0. 01 ） . Conclusions Transfeetion of the antisense MBD1 gene eukarvotie expression vector significantly redueed the expression level of MBDI gene in human eholangiocarcinoma cell line QBC-939, and suggestes that MBD1 gene plays animportant role in the development of cholangiocarcinoma and that transfection of antisense MBD1 gene may be a new method to treat cholangiioearcinoma.