摘要
AIM: To construct a gene modified hepatocellular carcinoma (HCC) specific EGFP expression vector regulated by abbreviated cis-acting element of AFP gene.METHODS: The minimal essential DNA segments of AFP gene enhancer and promoter were synthesized through PCR from Genome DNA of HepG2 cells. Gene fragments were then cloned into the multiple cloning site of non-promoter EGFP vector pEGFP-t. Recombinant plasmid was transferred into positive or negative AFP cell lines by means of lipofectamine. The expression of EGFP was tested by fluorescence microscope and flow cytometry. The effect of all-trans retinoic acid (ATRA) on the expression of EGFP was tested in different concentrations.RESULTS: By the methods of restriction digestion and sequence analyses we confirmed that the length, position and orientation of inserted genes of cis-acting element of AFP were all correct. The transcription of EGFP was under the control of AFP cis-acting element. The expressing EGFP can only been detected in AFP producing hepatoma cells.The expression rate of EGFP in G418 screened cell line was 34.9±4.1%. 48 h after adding 1×10-7M retinoic acid, EGFP expression rate was 14.7±3.5%. The activity of AFP gene promoter was significantly suppressed by addition of 1×10-7M retinoic acid (P＜0.05, P=0.003, t=6.488).CONCLUSION: This recombinant expression vector can be used as a gene therapy vector for HCC. The expression of tumor killing gene will be confined within the site of tumor and the activity of which can be regulated by retinoic acid.
AIM:To construct a gene modified hepatocellular carcinoma (HCC)specific EGFP expression vector regulated by abbreviated cis-acting element of AFP gene. METHODS:The minimal essential DNA segments of AFP gene enhancer and promoter were synthesized through PCR from Genome DNA of HepG2 cells.Gene fragments were then cloned into the multiple cloning site of non-promoter EGFP vector pEGFP-1.Recombinant plasmid was transferred into positive or negative AFP cell lines by means of lipofectamine.The expression of EGFP was tested by fluorescence microscope and flow cytometry.The effect of all-trans retinoic acid(ATRA)on the expression of EGFP was tested in different concentrations. RESULTS:By the methods of restriction digestion and sequence analyses we confirmed that the length,position and orientation of inserted genes of cis-acting element of AFP were all correct.The transcription of EGFP was under the control of AFP cis-acting element.The expressing EGFP can only been detected in AFP producing hepatoma cells. The expression rate of EGFP in G418 screened cell line was 34.9±4.1%.48 h after adding 1×10^(-7)M retinoic acid,EGFP expression rate was 14.7±3.5 %.The activity of AFP gene promoter was significantly suppressed by addition of 1×10^(-7)M retinoic acid(P<0.05,P=0.003,t=6.488). CONCLUSION:This recombinant expression vector can be used as a gene therapy vector for HCC.The expression of tumor killing gene will be confined within the site of tumor and the activity of which can be regulated by retinoic acid.
基金
Natural Scientific Foundation of China,No.30271474
