hTERT,CEA及CMV启动子在人结肠癌细胞株中的转录活性比较

Comparison of the transcriptional activity of hTERT, CEA and CMV promoter in human colon carcinoma cell lines

目的:比较人端粒酶催化亚单位(hTERT),癌胚抗原(CEA)及巨细胞病毒(CMV)启动子在人结肠癌细胞株LoVo和SW480中的转录活性。方法:设计引物应用PCR法从人结肠癌基因组中克隆hTERT和CEA启动子;用双酶切和PCR法切除原始载体pLVX-EGFP-3FLAG中的CMV启动子后,将hTERT,CEA启动子与该载体重组,构建出重组质粒pLVX-hTERTp-EGFP-3FLAG和pLVX-CEAp-EGFP-3FLAG;将上述两种质粒及原始载体(含CMV启动子)分别瞬时转染人结肠癌细胞株LoVo和SW480后,检测两种细胞株绿色荧光蛋白表达。结果:经PCR,酶切及测序鉴定,克隆及载体构建完全正确。CMV,hTERT及CEA启动子的转录活性(绿色荧光细胞数/总细胞数)在LoVo细胞中依次为54.7%,33.0%,9.5%;在SW480中依次为16.5%,10.1%,8.5%,差异均有统计学意义(均P<0.05)。结论:在人结肠癌细胞株中,转录活性以CMV启动子最高,hTERT启动子次之,CEA启动子最低。该结果可为结肠癌的靶向性基因治疗研究提供参考。

Objective： To compare the transcriptional activity of human telomerase reverse transcriptase （hTERT）, carcino embryonic antigen （CEA） and cytomegalovirus （CMV） promoters in human colon cancer LoVo and SW480 cells.Methods： After the primer sets were designed, the hTERT and CEA promoters were doned by PCR amplification from the genome of colon cancer cells. The CMV promoter was removed from the original vector pLVX-EGFP- 3FLAG by double digestion and PCR method, and the hTERT and CEA promoters were introduced into the vector to construct the recombinant plasmid pLVX-hTERTp-EGFP-3FLAG and pLVX-CEAp-EGFP-3FLAG. Colon cancer LoVo and SW480 cells were transiently transfected with the above two recombinant vectors and the original vector （containing CMV promoter） respectively, and the expressions of green fluorescent protein in the two cell lines were determined. Results： Results of PCR, enzyme digestion and sequencing showed that the cloning products and plasmid constructions were completely correct. The transcriptional activities （number of ceils expressing green fluorescence/total number of cells） of CMV, hTERT and CEA promoters in LoVo cells were 54.7%, 33.0% and 9.5%p and in SW480 cells were 16.5%, 10.1% and 8.5%, respectively. All the differences had statistical significance （allV〈0.0s）. Conclusion： In human colon cancer cells, the transcriptional activity of CMV promoter is the highest, hTERT promoter is second and CEA promoter is the lowest. These results may provide information for the study of targeted gene therapy of colon cancer.