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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 activities of human telomerase catalytic subunit (hTERT), carcinoembryonic antigen (CEA) and cytomegalovirus (CMV) promoter in human colon cancer cell lines LoVo and SW480. METHODS: Primers were designed to clone hTERT and CEA promoters from human colon cancer genome by PCR. The CMV promoter in the original vector pLVX-EGFP-3FLAG was excised by double enzyme digestion and PCR. The hTERT and CEA promoters The recombinant plasmids pLVX-hTERTp-EGFP-3FLAG and pLVX-CEAp-EGFP-3FLAG were constructed by recombination. The two plasmids and the original vector (including CMV promoter) were transiently transfected into human colon cancer cell lines LoVo and SW480 , Detected two kinds of cell line green fluorescent protein expression. Results: After PCR, restriction enzyme digestion and sequencing, cloning and vector construction were completely correct. The transcriptional activity (green fluorescent cells / total cell number) of CMV, hTERT and CEA promoters were 54.7%, 33.0% and 9.5% in LoVo cells and 16.5%, 10.1% and 8.5% in SW480, respectively All were statistically significant (P <0.05). CONCLUSION: In human colon cancer cell lines, the transcriptional activity of CMV promoter is highest, hTERT promoter is second, and CEA promoter is the lowest. The results provide a reference for the study of targeted gene therapy of colon cancer.