In Vitro Experiments Study Of Radiation Sensitizing Effect Of Adenovirus-thymidine Kinase Suicide Gene Prodrug System On CNE-2 Hunman Nasoparyngel Cafcionam Cell Line

Objective:In this study, used recombination adenovirus thymidine kinase(ADV-TK) that have into Phase II clinical trials to transfected CNE-2 nasopharyngeal carcinoma cells in vitro, observedADV-TK/GCV (Adenovirus-thymidine kinase/ganciclovir)suicide gene prodrug system for nasopharyngeal carcinoma CNE-2 cell-killing effect and radiosensitization.Methods:The nasopharyngeal carcinoma CNE-2 cells were transfected with different multiplicity of infection (Multiplicity of Infection, MOI)of ADV-TK,3h later, the medium be replacemented with fresh medium, cultured for 72 hours continuously,observed cell morphological changes before and after transfection under inverted microscope and cell survival rate and conditions that to be infected with different MOI was detected by WST-1.nasopharyngeal carcinoma CNE-2 cells were treated by different concentrations of GCV,cultured for 72 hours continuously, cells survival conditions that be treated with different concentrations of GCV were observed under inverted microscope,cells survival rate that be treated with different concentrations of GCV were detectde by WST-1.Selected the the maximum MOI=100 but no apparent toxic effects to transfect CNE-2 nasopharyngeal carcinoma cells as further experimental study,3h later,the medium be replacemented with fresh medium, cultured for 24h continuously, the cells were treated with different concentrations of GCV,cultured for 72h continuously,cell's survival conditions were observed under inverted microscope, and cell survival rate be deteted by WST-1, calculated IC10, IC50 of GCV.Select the MOI of 100, and IC10 for radiosensitization,experiments were divided radiation only, ADV-TK plus radiation, GCV plus radiation, ADV-TK/GCV plus radiation; Cells were transfected with 100 of MOI of ADV-TK,3h later, the medium be replacement with fresh medium, cultured for 24 hours continuously, adding GCVIC10, to culture 48h continuously, four groups were given 0,0.5,1,2,3,4,6,8, lOGyy ray irradiation,each irradiation dose group have three dishes, after irradiation, medium replaced with fresh medium immediately,cultured for 14d, observed CNE-2 cell killing effect of every group, Colony forming assay was used for survival fraction analysis and Single-hit multitarget model was used to plot survival curve,calculated radiosensitization ratio. Results:Observed under The inverted microscope,CNE-2 cell that be transfected with ADV-TK volume increase compared with the control group, outline more obscure, boundary less clear, there are more cells thick black granular material, mainly in the nucleus, but not ADV-TK gene transfected cells there is no obvious particulate matter.WST-1 test cell viability, when MOI were 100 and 1000, the survival rate was 98.95%,86.70%,respectively. When MOI≥1000,it was obvious toxicity for nasopharyngeal carcinoma CNE-2 cells.And when GCV concentration was 1000ug/ml, 100ug/ml, the survival rate of nasophary-ngeal carcinoma CNE-2 cells were 98.28% and 84.77%,when GCV concentration was 1000ug/ml cells obvious cytotoxicity.Select the MOI of 100 transfected nasopharyngeal carcinoma CNE-2cells, different concentrations of GCV treated cells, calculated IC10 and IC50,it was 7.1968ug/ml,196.0358ug/ml respectively.Radiosensitization experiments, the values of Do, Dq, and N of CNE-2 cells received radiation treatment alone were 1.238Gy,2.838Gy,3.284 respectively.Those of CNE-2 cells received ADV-TK plus radiation were 1.237Gy,2.607Gy,and 3.108 respectively, SERDo, SERDq and SERSF2 were 1.001,1.089 and 1.036 respectively.Those of CNE-2 cells received GCV plus radiation were 1.232Gy 2.800Gy,and3.274 respectively, SERDo, SERDq and SERSF2 were 1.005,1.014 and 1.005 respectively.Those of CNE-2 cells received ADV-TK/GCV plus radiation were 0.800Gy 1.561Gy,and2.775 respectively, SERDo, SERDq and SERSF2 were 1.407,1.818 and 2.414 respectively. Conclusions:(1)ADV-TK successfully transfected CNE-2 cells.(2)It have obvious toxicity for CNE-2 cells when ADV-TK (MOI)≥1000(3) GCV≥1000ug/ml on nasopharyngeal carcinoma CNE-2 cells was significantly inhibited.(4) ADV-TK/GCV suicide gene prodrug system for nasopharyngeal carcinoma CEN-2 cell killing effect was significantly and concentration-dependent with GCV.(5) ADV-TK/GCV suicide gene prodrug system on NPC CEN-2 cells was radiosensitization.