In Vitro Experimental Study Of HSV1-TK/GCV Suicide Gene Therapy For Hepatocellular Carcinoma Mediated By Ultrasound Contrast Agent

The advent and development of gene therapy has provided new insights into the treatment for liver cancer. Being a"hot spot"of biomedical researches, gene therapy, especially that mediated by HSV1-TK/GCV suicide gene system, has now become one of the most effective approaches for the treatment of liver cancer and attracted more and more attentions. The key problem of gene therapy is to choose a reliable and effective vector for transfecting gene of interest into the target cell. However, existing viral or non-viral vectors have drawbacks, such as hepatic toxicity, low tissue specificity and lack of security, which limit their clinical appliance.Recent studies have showed that ultrasound-targeted microbubble destruction (UTMD) were able to increase membrane permeability and enhance exogenous genetic materials into targeted cells by mechanical and cavitation effects of UTMD, ultrasound-targeted microbubble destruction is a new noninvasive gene transfer technology, providing a new method of gene therapy for liver cancer. In the current study, we prepare to construct vector pIRES2-EGFP-TK that co-expressing enhanced green fluorescent protein and (EGFP) and herpes simplex virus type 1 thymidine kinase (HSV1-TK). And by transfecting the recombinant plasmid into the HepG2 cell line by ultrasound-targeted microbubble destruction, we seek to observe the expression of the EGFP and TK protein in hepatocarcinoma cells and .explore the specific lethal effectiveness of treating hepatoma carcinoma in vitro using ultrasound-targeted microbubble destruction mediated by HSV1-TK/GCV suicide gene system. Our study can provide reliable experimental evidences for the research of targeted gene therapy for hepatoma carcinoma. The study includes the follwiong two parts:PARTâ… :CONSTRUCTION OF A VECTOR CO-EXPRESSING EGFP AND HSV1-TK AND ITS EXPRESSION IN HUMAN HEPATOMA CELL LINE HEPG2Objective To construct a vector co-expressing EGFP and HSV1-TK, and detect its expression level in human hepatoma cell line HepG2.The insertion and the direction of the insert were confirmed by polymerase chain reaction (PCR) and restriction enzyme digestion. Methods HSV1-TK gene was isolated from pORF-HSV1tk plasmids and cloned into eukaryotic expression plasmid pIRES2-EGFP. The recombinant plasmid pIRES2-EGFP-TK was identified by restriction endonuclease digestion and DNA sequencing. Then recombinant plasmid was transfected into hepatoma carcinoma cell line HepG2 by liposome reagent,and the expression of EFGP were observed by fluorescence microscopy in cells , TK protein was analyzed by Western blotting . Results The sequence of the cloned DNA fragment was identical to HSV1-TK that was reported on GeneBank and direction of the insert were correct. The recombinant expression plasmid was successfully transfected into HepG2 cell line, and green fluorescent was observed under fluorescent microscope.The protein expression level of TK was high. Conclusion The recombinant eukaryotic co-expression vector of EGFP and HSV1-TK was successfully constructed and can be effectively expressed in HepG2 cell line.PARTâ…¡: HSV1-TK/GCV SUICIDE GENE SYSTEM FOR HEPATOCELLULAR CARCINOMA BY ULTRASOUND CONTRAST AGENT MEDIATED IN VITROObjective To explore the specific lethal effectiveness of treating hepatoma carcinoma in vitro using ultrasound-targeted microbubble destruction mediated by HSV1-TK/GCV suicide gene system; and indentify the effects of ultrasound-targeted microbubble destruction treatment on the cell viability and provide experimental basis for further research. Methods HepG2 was seeded in the 24-well plates, and randomly divided into 5 groups, (1) Blank control group. (2) HSV1-TK: add pIRES2-EGFP-TK only ;(3) HSV1-TK+UM: add gene and ultrasound contrast agent but didn't exposed to pulsed-ultrasound; (4) HSV1-TK+US: add gene and ultrasound contrast agent then exposed to pulsed-ultrasound; (5) HSV1-TK+UM+US: add gene and ultrasound contrast agent and pIRES2-EGFP-TK, then exposed to pulsed-ultrasound. After 24 hours, expression of EGFP was observed on fluorescent microscopy and quantified by FACS analysis. Western Blotting was used to detect the expression of TK protein. 24 hours after transfection, different concentrations of GCV were added into the culture mediums, then cell were cultured for another 72 hours. The viability of HepG2 was measured by MTT assay. Results Gene fluorescence intensity and tansfection efficiency in group of ultrasound contrast agent and ultrasound and pIRES2-EGFP-TK were higher than other groups, TK protein in the fifth group was higher than other groups(P<0.05).The cell viability had no significant difference before add GCV. Almost all HepG2 in the fifth group have been killed when the concentration of GCV is above 50ug/mL, and we observed almost no killing effect in other groups after add different concentrations of GCV. Conclusion Ultrasound-targeted microbubble destruction can enhance the efficiency of gene transfection without obvious damage to cell viability in HepG2.And the method can also enhance the killing effect of HSV1-TK/GCV suicide gene system in vitro. This experimental study provides a good basis for the research of targeted gene therapy for hepatoma carcinoma.