摘要
目的 分析DNA甲基化酶 1(DNMT1)基因的真核表达质粒对人结肠癌细胞肿瘤相关基因的表达影响。方法 分别构建并转染含有人正义和反义DNMT1的真核表达质粒入结肠癌SW 1116细胞 ,PCR和限制性内切酶证实转染结果 ,以Western印迹法分析各组细胞DNMT1蛋白的表达情况。定量PCR检测hMLH1、hMSH2及c myc、p16 INK4A 基因的表达。结果 经G4 18筛选得到稳定转染DNMT1基因的结肠癌细胞系 ,且分别在该转染有正义和反义质粒的细胞系中 ,DNMT1蛋白表达上调和下调。同时发现转染正义DNMT1的细胞中hMLH1、hMSH2及c myc的表达降低 ,而转染反义DNMT1的细胞中hMSH2的表达明显增强。各组细胞p16 INK4A基因的表达差异不明显。
Objective To analyze the effect of eukaryotic expression vector containing sense and antisense DNA methyltransferase (DNMT1) gene on the transcript level of tumor associated genes in human colon cancer cell line. Methods Recombinant plasmid, including sense DNMT1 (HMT) and antisense DNMT1 (THM) gene, were constructed and transfected into SW1116 cell by using the lipofectamine. Then G418 filtration was performed. The expression of DNMT1 protein was examined by Western blotting. The transcription level of hMLH1, hMSH2, c myc and p16 INK4A genes were detected by RT PCR. Results The sense and antisense eukaryotic expression vectors were successfully constructed and then the constructed recombinant plasmids were transfected into SW1116 cell. The protein levels of DNMT1 have been up regulated and down regulated in SW1116 cells transfected with HMT and THM plasmids, respectively. The mRNA level of hMLH1, hMSH2, c myc gene were down regulated in the sense DNMT1 transfected cell. The mRNA level of hMSH2 was up regulated in the antisense DNMT1 transfected cell. However, the transcription level of p16 INK4A gene could not be associated with DNMT1 in SW1116 cell.Conclusion DNMT1 can regulate the expression of the tumor associated genes in human colon cancer cell line SW1116.
出处
《中华消化杂志》
CAS
CSCD
北大核心
2004年第2期67-70,共4页
Chinese Journal of Digestion
基金
国家自然科学基金 (3 0 170 413 )
