The Construction And Research Of Radio-sensitivity Tumor Suicide Gene Vector For Uterine Cervical Cancer

Objective Recombinant plasmid vector pcDNA3.1-CMV-Egr-1-CDTK was constructed by fusing of early growth respons-1(Egr-1) promoter to the double suicide gene CDTK gene; To transfer human cervical cancer cell lines (Hela) with recombinant plasmid vector pcDNA3.1-CMV-Egr-1-CDTK,after irradiated by ⁶⁰Co-γ rays ,the CDTK mRNA expression in the transfected cell line and the killing effects and the by-stander killing effects in vitro of double suicide genes on the cell lines were observed; The animal model of Balb/c nude mice was established by injected subcutaneously with exponentially growing transfected cell lines for further research of the killing effect on cervical cancer in vivo by radio-induced suicide gene after exposure to ⁶⁰Co-γ rays.Methods The CDTk gene were amplified with polymerase chain reaction (PCR) technique and were fused into CDTK gene, linked with plasmid vector pcDNA3.1, Egr-1 promotor and CMV enhancer were amplified and then inserted CMV enhance infront of Egr-1, aquired plasmid vector pcDNA3.1-CMV-Egr-1-CDTK. Which were transfected into cervical cancer cell lines with electroblot as a delivery system. After accepted with ⁶⁰Co-r rays irradiation, prodrugs(5-FC,GCV) was used in the cells ,RT-PCR, Western blot analysis were used to determine the CDTK mRNA expression in Hela cells which had been transfected by pcDNA3.1-CMV-Egr-1-CDTK plasmid vector. The viabilitys of cells were determined by the method of MTT .For the purpose ,we can research the killing effect and the by-stander killing effect of radio-induced double suicide gene on the cervical cancer cell lines in vitro and the sensitization to irradiation. The Balb/c nude mice were injected subcutaneously with pcDNA3.1-CMV-Egr-1-CDTK. The tumorigenesis characteristic were obtained. The animals were devided into five groups and after the therapy of irradiation and prodrugs respectively, the volume of tumor were observed. Furthermore, the pathological results of tumor was observed. At the same time, RT-PCR was used to detect the tumors CDTK amplification fragements.Results The successful construction of recombinant plasmid vector was certificated through enzyme cutting and sequencing. The length of Hela/CDTK gene was certificated by RT-PCR and Western blot, a 59KD protein was obtaind which was equal to the expection of CDTK gene sequencing. So proved the recombinant plasmid...