Experimental Study Of Low Frequency Ultrasound Combined With HSV1-TK/GCV Gene Containing Microbub Bles Suicide Gene To Inhibit Residual Hepatocellular Carcinoma After HIFU Ablation

BACKGROUNDPrimary hepatocellular carcinoma is one of the most common malignancies.Surgical resection of cancer and liver transplantation is an effective method for the treatment of early liver cancer at present,but most patients have been in the advanced stage of treatment,so open up a new way of treatment is the current hot spots of liver cancer research.High-intensity focused ultrasound(HIFU)can focus low-energy ultrasound on the tumor target area to produce transient high temperature,cavitation effect,thereby kill the cancer cells within the focal domain.The effect of HIFU is obvious,but there is incomplete due to ablation and easy to recur after surgery and other issues.Exploring a new adjuvant therapy is the direction of research field in recent years.Herpes simplex virus type I thymidine kinase / ganciclovir(HSV1-TK / GCV)suicide gene has a good effect on killing cancer cells.Therefore our aim of this study is to investigate the inhibitory effect of cationic microbubbles combined with low frequency ultrasound(HSV1-TK / GCV)on residual hepatocarcinoma SMCC-7721 cells after incomplete ablation of HIFU.OBJECTIVETo investigate the inhibitory effect of low intensity ultrasound combined with HSV1-TK/GCV gene carried by cationic microbubbles on high-intensity focused ultrasound incompleted ablation of hepatocellular carcinoma cellsMETHODSThe cationic microbubbles were prepared by membrane hydration method and the microbubbles were connected with the plasmids according to the principle of electrostatic adsorption.The HIFU was fixed to one appropriate power,and irradiated at the different time.The optimal irradiation time was selected according to the cell survival state.SMCC-7721 cells were irradiated with HIFU to select the best residual hepatocellular carcinoma cell subline for stable preservation.Cationic microbubbles loading TK gene were transfected by low intensity ultrasonic radiating.And then they were divided into control group(residual cancer),residual cancer+TK/GCV group,residual cancer+ultrasound(US)+TK/GCV group and residual cancer+microbubbles+ US+TK/GCV group.Transfection and expression were detected by Western Blot.Flow cytometry was used to observe and detect the gene transfection efficiency.The suitable concentration of GCV was selected,and the survival rate of cells in the same condition of each group was detected.RESULTSThe size of microbubbles was uniform,and the morphology and dispersion were good.The size of microbubbles was abount(542.80±24)nm,zeta was abount(40.13±1.50)mV.The preparation of nano-sized cationic microbubbles can be combined with weakly negatively charged plasmid and pass through the vascular endothelium.The gene carrying capacity of 3.6 g/108 microbubbles,and the gene binding rate was(32.18±4.26)%.HIFU power was 5 W,irradiated for 40 s residual cancer model was established.The optimal concentration of GCV was 100 ?g/ml.In the same conditions,the cell survival rate of residual cancer+micro bubble +US+TK group was the lowest([56.84±3.18]%),and the expression of TK protein was the hightest.The difference was statistically significant(all P<0.05).CONCLUSIONLow frequency ultrasound combined with HSV1-TK/GCV suicide gene cationic microbubble has a good inhibitory effect on SMCC-7721 cells after ablation of HIFU.