Investigation Of The Radiosensitive And Tumor-specific Suicide Gene Vector Of Gene Therapy For Nasopharyngeal Carcinoma

Objective: Nasopharyngeal carcinoma (NPC) is one of the common malignant tumors in China. Its age of onset is much earlier than other tumors', mostly about 30-50 year old. The disease is far more common in south China than in other place else. It is reported that of the total incidence of NPC is account for 18-30%, while 78% in head and neck cancers. Now the radiotherapy and chemotherapy are the main therapy methods for NPC. To enhance the specific antitumor effect, novel vectors with radiosensitivity and specificity of tumor were constructed in this study, which can degrade the dose of ray and chemotherapeutic medicine using its double killing effect. Methods: The Egr-1 promoter, CD gene, hTERT promoter, Survivin antisense oligonucleotides were amplified. And then all the fragments were digested and ligated to the pcDNA3.1, creating pcDNA3.1-Egr-CD(pEC),pcDNA3.1-hTERT-Survivi n(pTS),pcDNA3.1-Egr-CD-hTERT-Survivin(pEC-TS) expressing vectors. After transfer of the CNE-2 cells and radiographic exposure, CD and Survivin gene expression in CNE-2 cells were detected by RT-PCR. HPLC was employed to determine the transformation from the prodrugs 5-FC to 5-FU. And then the apoptosis of all groups was indicated by Hoechest33258 nucleus staining. Meanwhile, the cell survivability after treatment was detected by MTT assay. Finally, the animal model was constructed to analyse the antitumor effect of different vectors in vivo.Results: The vectors were verified by enzymes digestion and sequencing. And the results that GFP expression regulated by hTERT promoter was different among various cell lines revealed the specificity of hTERT promoter. After the transferred cells treated with radiation and prodrugs, the specific fragments of mRNA were detected by RT-PCR. And a high concentration of 5-FU was determined using HPLC. The Survivin gene expression was significantly suppressed after the Survivin antisense oligonucleotides were transferred to the CNE-2 cells. Hoechest33258 nucleus staining results showed in pEC-TS group, a large scale cells were dead with strong blue light stimulated by the emission of radiation in condensed chromatins. While MTT assay revealed a similar result, the cell survivability in pEC-TS group was the lowest, which showed an significant different in antitumor effect compared with other treatment groups (p＜0.05). The in vivo results correpondingly showed an obvious tumor regression in pEC-TS group after a consecutive treatments with prodrugs and radiation. The tumor volumes of each group have significant difference according to the tumor growth curve (p＜0.05). Finaly the pathology results showed a large number of cells died in tumor tissues with nucleus disappeared. Conclusions: After pEC-TS was transferred to the CNE-2 cells, under the control of Egr-1 promoter the CD gene expressed after radiation was administrated. 5-FC could be converted to 5-FU efficiently, and as well as the Survivin gene expression was degraded specificly by hTERT promoter, resulting in the rise of sensitivity to 5-FU for CNE-2 cells. Therefore this study provides new experimental basis for gene therapy for NPC.