Construction And Killing Effects Of Cytosine Deaminase And Thymidine Kinase Fusion Suicide Gene Vector On Retinoblastoma Y79 Cells

Retinoblastoma is the most common primary intraocular malignancy of childhood.It is a big threat to eyesight and lifespan for children. Currently,traditional treatments for retinoblastoma are limited or toxic potentially.With the development of molecular biology of tumor,people pay much attention to the gene therapy.At present,the suicide gene therapy for malignancy is the research that is hot all over the world.In our research,we established a vector that contains yeast cytosine deaminase(yCD)and herpes simplex type 1 thymidine kinase(TK)fusion suicide gene driven by hTERT promoter(pcDNA3.1-CMV-hTERT-CDTK, pc-ChCDTK).And then,we investigated the killing effects of the vector on human retinoblastoma Y79 cells in vitro and in vivo.PartⅠConstruction of Tumor Cells Specific Expression Vector of Suicide Gene of CDglyTK Driven by hTERT Promoter[Objective]To construct the tumor cells specific vector of suicide gene of CDglyTK driven by hTERT promoter.[Methods]The CMV enhancer,hTERT promoter,yCD and TKgly gene were amplified with polymerase chain reaction(PCR) technique.The transient vectors,T-CMV,T-hTERT,T-yCD,T-TKgly,were established.These fragment were inserted into plasmid vector pcDNA3.1 and aquired plasmid vector pcDNA3.1-CMV- hTERT - CDTK (pc-ChCDTK)The vector was identified by enzyme cutting and sequencing.[Results]We got 6773bp and 288bp fragments through enzyme cutting.The result of sequencing was equal to the expection.[Conclusions]The vector that contains CDTK fusion gene was constructed successfully PartⅡKilling Effects of Tumor Cells Specific Vector of Suicide Gene of CDglyTK Driven by hTERT Promoter on Y79 Cells in Vitro[Objective]To investigate the killing effects of Tumor Cells Specific Vector of Suicide Gene of CDglyTK Driven by hTERT Promoter on Y79 Cells in Vitro.[Methods]At first,the recombinant plasmid was transfered into Y79 cells with electroblot as a delivery system.RT-PCR and Western blot analysis were used to determine the CDTK mRNA and protein expression in Y79 cells which had been transfected by pcDNA3.1-CMV- hTERT -CDTK plasmid vector.The conversion efficiency of 5-Fc into 5-FU in yCDglyTK-expressing Y79 cells was measured by HPLC.The killing effects of double suicide genes on retinoblastoma Y79 cells that treated with 5-Fc,GCV of different concentration were determined by the method of MTT.[Results]The expression of CDTK gene in Y79 cells was certificated by RT-PCR and Western blotting and a 59KD protein was obtaind which was equal to the expection of CDTK gene sequence.In MTT analysis,there were significant difference between transfected and non-transfected survival Y79 cells(P＜0.05).The survival Y79 cells treated with 5-Fc and GCV was lower than 5-Fc or GCV.[Conclusions[The recombinant plasmid vector pcDNA3.1-CMV-hTERT-CDTK can be transcribed and translated into CDTK fusion protein in Y79 cells.The transfer of the CDglyTK fusion gene into retinoblastoma Y79 cells followed by the administration of 5-FC or GCV can kill Y79 cells in vitro.The killing effect of two predrugs was stronger than one predrug. PartⅢKilling Effects of Tumor Cells Specific Vector of Suicide Gene of CDglyTK Driven by hTERT Promoter on nude mice modle of retinoblastoma in vivo[Objective]To investigate the killing effects of Tumor Cells Specific Vector of Suicide Gene of CDglyTK Driven by hTERT Promoter on retinoblastoma in vivo.[Methods]The BALB/C nude mice were devided into two groups,ten mice each.The experimental mice were injected subcutaneously with the CDTK expressional retinoblastoma Y79 cells. The control mice were injected with non-transfected Y79 cells.Intraperitoneal injections with 5-FC and GCV were started 7 days after tumor injection,to allow the tumor time to grow and become established.All the mice with tumor received prodrug once per day for 15 days.the volume of tumor were observed.Furthermore,the pathological results of tumor was observed.At the same time,RT-PCR was used to detect CDTK amplification fragements in the tumors.[Results]The volume of the tumor in experimental group, compare to the control group,showed a significant decrease.There was significant difference in the tumor volume between the two groups(P＜0.05).There was generous necrosis in the transfected group.The result of RT-PCR showed 403bp fragment that was eaqual to the expection.[Conclusions]Tumor Cells Specific Vector of Suicide Gene of CDglyTK and prodrugs can inhibit the growth of tumor of Y79 subcuneously.