Experimental Study On The Inhibitory Effect Of HSV-TK/GCV Suicide Gene System Mediated By Ultrasound Cationic Nanobubbles On Gastric Cancer Cells

Objective:To observe the effects of herpes simplex virus-thymidine kinase(HSV-TK)gene transfection in vitro and combined with ganciclovir(GCV)on the proliferation,apoptosis,cycle,invasion and migration of human gastric cancer NCI-N87 cells,and to explore the transfection ability of HSV-TK/GCV system mediated by ultrasound cationic nanobubbles and its inhibitory effect on gastric cancer cells.Methods:Gastric cancer NCI-N87 cells were irradiated with different ultrasound intensities(0.5 W/cm~2,1.0 W/cm~2,1.5 W/cm~2,2.0 W/cm~2)and different time(15 s,30s,45 s,60 s).The optimal ultrasound parameters were screened by MTT assay.The cationic nanobubbles carrying HSV-TK gene were transfected using the optimized ultrasound parameters described above.The experiment was divided into 5 groups:negative control group,simple plasmid TK group,cationic nanobubble+plasmid TK group,ultrasound+plasmid TK group,ultrasound+cationic nanobubble+plasmid TK group.The expression of TK protein was observed by Western Blot.The concentration of GCV without significant inhibition of cell viability was screened by MTT assay.Flow cytometry was used to detect the cycle and apoptosis rate of gastric cancer NCI-N87 cells in each group after GCV treatment.The killing effect of HSV-TK/GCV suicide gene system on gastric cancer NCI-N87 cells was observed by MTT assay.Transwell assay was used to detect the invasion and migration ability of gastric cancer NCI-N87 cells in each group.Results:The MTT method showed no significant inhibition of cell growth and good cell activity when the ultrasound intensity was 1.0 W/cm~2,frequency was 1 MHz and irradiation time was 30 s.Therefore,this parameter was selected as the optimal irradiation condition for the study of nanobubble-carrying gene transfection;and there was no significant difference in the effect of different transfection methods on cell viability;the expression of TK protein was significantly higher in the ultrasound+cationic nanobubble+TK group than in other experimental groups(P<0.01).MTT results showed that the survival rate of NCI-N87 cells was≥95%at GCV concentrations≤100μg/ml,and cell viability was not significantly affected,and the difference in cell survival rate was statistically significant compared with the concentration of 1000μg/ml(P<0.01),so the optimal concentration of GCV was selected as 100μg/ml;the MTT results showed that the cells transfected with HSV-TK gene had a significant killing effect and bystander effect after GCV treatment;Flow cytometric analysis indicated that the majority of cells were blocked in the S-phase of ultrasonic+cationic nanobubble+TK group,and the cell growth was inhibited obviously;Flow cytometry showed that the apoptosis rate of cells in the ultrasound+cationic nanobubble+TK group was significantly higher than that of other experimental groups(P<0.01);Transwell assay showed that the number of cell invasion and migration in the ultrasound+cationic nanobubble+TK group was significantly lower than that in the other experimental groups(P<0.01).Conclusion:Under the optimal ultrasound parameters,ultrasound-mediated cationic nanobubbles can promote HSV-TK gene transfection and effective expression in cells;after combined with prodrug GCV,ultrasound-mediated cationic nanobubbles can enhance the effect of HSV-TK/GCV system on inhibiting proliferation and inducing apoptosis of gastric cancer NCI-N87 cells,inhibit cell invasion and migration,and block cell cycle,thus exerting anti-tumor effect.This study provides an experimental basis for gene therapy of gastric cancer.