Radiosensitization By Double Suicide Gene Systerm In ACC-2 Cell

Objective Adenoid cystic carcinoma,which is also called cylindroma,was first reported by Billroth.Most scholar considered that it comes from salivary ducts,or probably the oral squamous basal cells.It characterizes by encroaching the nerve and far matastasis.The treatment of adenoid cystic carcinoma gives priority to operation combined with post operation irradiation and chemistry therapy,but the curative effect is still dissatisfactory.Therefore,it makes sense to explore new therapy.Recently,the gene therapy of malignat tumor is developed rapidly and there are many methods about the gene therapy that include:suicide gene therapy,immunologic gene therapy,drug resistance gene therapy, angiostatin gene therapy and so on.The sucide gene therapy is one of the most potential approach of antitumor.Many study had investigated that the double suicide gene therapy was much better than the suicide gene therapy.The study by Wang Anxun had also revealed that the system of HSV-TK/GCV had radiosensitivity and could increased the radiotherapy sensitivity of squamous cell carcinoma.In this study,We constructed eukaryotic expression plasmids pIRES-CD and pIRES-TK,and transfected them into ACC-2 cells by electroporation.The prodrug GCV and 5-FC were used in order to observe the radiosensitization by CD and TK double suicide gene therapy in ACC-2 under the aerobic and anoxic condition and to find more effective suicide gene therapy.Methods The eukaryotic expression plasmids pIRES-CD and pIRES-TK were introduced into ACC-2 cells by electroporation.Then ACC-2 cells stably expressing CD and TK gene were obtained by 10-days positive select with 400μg/ml G418.Total RNA was extracted and the expression of the CD and TK gene in transfected ACC-2 cells was identified by RT-PCR.The positive transfected ACC-2 cells were treated with radiotherapy of different dose(0,2,4,6,8,10Gy) and prodrug system in aerobic and anoxic condition.Then through cell clone formation assay to study the radiosensitization by CD-TK double suicide gene therapy and prodrug system in ACC-2.The data was analyzed by multiple factor ANOVA of SPSS13.0 software packet.Results RT-PCR analysis demonstrated that CD and TK gene was effectively expressed in ACC-2 cells.With the increased of X-ray dose, the colony forming rate dropped significantly after radiotherapy.In aerobic condition,the survival fraction of group ACC-2/CD-TK+prodrug were greatly lower than that of group ACC-2and groupACC-2/CD-TK with the same dose(P＜0.05).In anoxic condition,the survival fraction of group ACC-2/CD-TK+prodrug was greatly lower than that of experiment groupACC-2 and groupACC-2/CD-TK with the same dose(P＜0.05).The colony forming rate in aerobic condition was greatly lower than that in anoxic condition of the same cells group and dose.Conclusions 1.The pIRES-CD and pIRES-TK were transferred into ACC-2 cells through electroporation.228bp and 361bp positive band were seen through RT-PCR,which showed that suicide gene could be transcribed and expressed in gene-transferred ACC-2 cells.2.The CD and TK double suicide gene therapy and prodrug system can enhance the radiosensitivity and the killing effect of ACC-2 cells in aerobic condition..And the effect was significant than the single suicide gene therapy in aerobic condition.3.The CD and TK double suicide gene therapy and prodrug system can enhance the radiosensitivity and the killing effect of ACC-2 cells in anoxic condition..And the effect was significant than the single suicide gene therapy in anoxic condition.4.In the same doses,the radiosensitivity of the same cell group under the anoxic condition was poorer than that of the cell anoxic condition。The consequence was conformity to the study of molecular biology.