Effects Of Adenovirus-mediated HSV-tk/GCV System On Lens Epithelium

Objective The efficiency of adenovirus-mediated herps simplex virus thymidine kinase/ganciclovir(HSV-tk/GCV) suicide gene therapy sysytem on calf lens epithelial cells was researched in vitro.To observe the phenomena of cell apoptosis and necrosis induced by HSV-tk/GCV therapy system and initially testify the mechanism of cell death.Methods The adenoviral vectors were propagated ,purified and titrated in 293 cell lines.Bovine lens epithelial cells were infected in vitro by the recombant adenoviral vector containing HSV-tk gene(suicide gene) and followed by the treatment of GCV.The killing efficiency of HSV-tk/GCV system on LECs(lens epithelial cells) exposure to various dosages of GCV(0-250ug/ml) and in different time(l-7days) was determined using the ways of MTT Colorimetric Assay and Trypan Blue Exclusion .As follows the bystander effect of HSV-tk/GCV system was testified. Some other experiments were done to see whether the cell infected time(l-8h) and vims MOI(multiplicity of infection) (O.l-lOOO)had the influnence on the killing efficiency or not.The E, deleted empty adenoviral vector was as the control. In addition ,the phenomena of cell necrosis and apoptosis was observed by scan electronic microscope,TUNEL(terminal-deoxynucleotidyl transferase mediated nickend labeling) and AO-EB(Acridine Orange-Ethidium Bromide) dyeness.At the same time to detect the levels of LECs apoptosis and necrosis and compare the difference between the experiment group and the control group.Results Bovine lens epithelial cells transinfected by adenoviral vector containing HSV-tk gene were significantly inhibited in vitro by GCV treatment.And the phenomenon of bystander effect was confirmed.By comparison there was no obvious toxin on the cells infected by empty adenoviral vector. Under the treatment of various dosages of GCV(0-250ug/ml), a significant difference of the cell survival rate existed between the ADV/HSV-tk group and the ADV/Empty group (/=4.94rP<0.01) .And also with the increase of the dosage of GCV,the cell survival rates in the two groups were gradually decreased.When the dosage of GCV is 25ug/ml in the ADV/HSV-tk group,only 21.3% of LECs were left.The killing efficiency of GCV increased with the prolonged time.About 54.7% of LECs were dead at the 1st day ,however ,till the 7th day only 8.2% of cells have survived. And the cell survival rate of the ADV/HSV-tk group was much more lower than that of the ADV/Empty group (t=\ 1.275rP<0.001). The experiment have confirmed the bystander effect ,when the transfected LECs occupied 10% of total LECs,85.7% of LECs were killed by GCV.According to the comparasion of cell survival rates at various cell infected time ,we found that adenoviral vectors could transinfect the LECs even within short 1 hour. We also knew that there were obvious effects of viral MOI on the cell survival rate. With the increase of MOI,the cell survival rate decreased. Through scan electronic microscope ,TUNEL and AO-EB dyeness ,we have observed some apoptosis LECs and large number of necrosis LECs in the two groups.The levels of LECs necrosis (f=14.716,P<0.001)and apoptosis (f=7.28,P<0.01) in the ADV/HSV-tk group were greatly higher than those in the group of ADV/Empty under AO-EB dyeness.Conclusions Adenoviral vector may successfully transduct HSV-tk gene(suicide gene)into bovine lens epithelial cells.The lens epithelium transfected by ADV/HSV-tk could be effectly killed by GCV.Through the observation of LECs apoptosis and necrosis caused by HSV-tk/GCV system,we might make the decision that the mechanism of cell death was probably necrosis and apoptosis. Therefore adenovirus-mediated HSV-tk/GCV suicide gene therapy system may provide an effective approach of posterior capsule opacification(PCO).