| Nasopharyngeal carcinoma(NPC) is a common malignant tumor in South ChinaQncluding Guangdong, Guangxi, Hunan and Fujian province etc). Among the pathological patterns of NPC, more than 90% are lowly-differentiated squamous carcinomas, so the primary treating method lies in radiotherapy. It is reported that the 5-year survival rate of early NPC patients treated with radio therapy was around 40%-50%. For those who get radiation for the second time, though suffering from the great damages by radiation, only one quarter show improvements and the 5-year survival rate drops to 12%-23%. Furthermore, almost all of the tumor cells may lose their sensitivity to radiotherapy-during the third radiation. Therefore, it is of great clinical significance to explore new ways to enhance the sensitivity of NPC cells to radiation.For a long time, many strategies including single stereotactic radiotherapy, stereoconformalradiotherapy, unconventionality fractionated radiotherapy and radio-sensitivitors such as high bressure oxygen, Hyperthermia, Bleomycin and nitromidazole have been tried.However.no matter what is used, the sensitivity of NPC to radiotherapy can' t be distinctly enhanced. More recently, the combination of gene therapy and radiotherapy has become a novel approach.In this study, using the inducibility of Egr-1 promoter, both in vitro and in vivo gene-radiotherapy of NPC were performed for the first time internationally.Egr-1 gene encodes 533 amino acids of nucleic acid transcription factor,and the promoter of Egr-1 gene contains 6 higly conservative domains CC(A+T)6GG induced by alive oxygen medium produced by radiation .which distinctly increase the expression of the downsdream gene controlled by Egr-1 promoter. So, compared with traditional therapies, NPC CNE-2 cells can be killed by irradiation and suicide gene/prodrug , at the same time , using the radio-inducibility of Egr-1 promoter the targeted gene therapy can be achieved, because the well-developed stereotactic radiotherapy can make it sure that radio-rays be accurately focused on tumor and the doses of irradiation to different tissues be effectively controlled. Thereby, the targeted gene therapy of NPC can be realized through the targeted and controlled irradiation, which has important significance for the improvement of the effect of therapy to NPC.In the study, we choose the confusion gene CD/UPRT cloned from Yeast as the downstream gene controlled by Egr-1 promoter for it' s excellent antitumor characteristic. It combines functions of CD gene and UPRT gene , therefore, converts 5-FC to 5-FU more effectively than CD gene. It is well to known that 5-FU can kill tumor cells and increase the inducibility of tumou cells to irradiation. Nowadays, there are only few reports in this field, in which the CD/UPRT confusion gene was mostly from E. coli. Since it was demonstrated that CD gene from Yeast has more advantage over that fromE. coli in therapeutic effect , I presume that CD/UPRT gene from Yeast has stronger antitumor effects than that from E. coli.Therefore, recombinant gene of pcDNA3.1(-)CMV. Egr-lCD/UPRT containing CD/UPRT gene controlled by Egr-1 promoter was constructed using the technique of molecular cloning and DNA recombination. In the rationale behind the vector selection, pcDNAS. 1(-)CMV was chose for the following reasons: (1) it is safe and free of immunogenicity; (2) the operation is easy enough because the plasmid is only 5. 4kb and all the insertion fragments of suicide genes in this research are less than 2.5 kb; (3) It is auniversally accepted notion that plasmid vector is the best vector for the insertion sequences less than 10kb. CMV promoter was isolated from cytomegalovirus which is a powerful initiator for the expression of the foreign genes. In the study, the enhancer of CMV promoter and Egr-1 promoter were respectively cloned from plasmid PEGFP-N1 and gDNA of the mouse, and the sequence analysis verified the sequence of the Egr-1 promoter from - 419 to +26. Then CMV. Egr-1fusion promoter of 893 bp was c...
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