After that directionally connect HSV-TK target gene fragment and pIRES2-EGFP (Invitrogen, USA) vect with the help of DNA ligase to obtain recombinant plasmid pIRES2-EGFP-TK. strategy for gene therapy in liver cancer. == Intro == Hepatocellular carcinoma (HCC) is one of the malignant tumors with high incidence around the world [1,2]. More than one million fresh instances appeared each year, particularly in the Asia-Pacific region. This disease offers rapid progress, high recurrence rate and traditional treatments have limited. With the continuous development of molecular biology, gene therapy for liver tumor has become a study hotspot and direction [3]. However, the security of viral vector, ineffectiveness of non-viral gene vectors and additional problems limit its further development [4,5]. Consequently, the search for an efficient, well focusing on and safe gene transfection system for malignancy gene therapy has become a focus of reseachers inteset. Recently a large number of studies have shown that ultrasound-targeted microbubble damage is a safe, effective, non-invasive, and physical gene transfection technology, which brings a new hope for gene therapy in liver cancer [6-8]. Ultrasound microbubbles mostly consist of gas [9]. The composition of its shell may include albumin, lipids, saccharide, non-ionic surfactants, polymers and additional materials [10]. At present the size has been developed to nano-scale and it has the ability to penetrate the vascular endothelium [11]. Microbubbles comprising gas will become compressed and expansed under the action of ultrasound with DMP 777 a certain intensity and rate of recurrence. When the sound energy reaches particular intensity, the microbubbles are immediately crushed. This will produce cavitation effect and mechanical effect to increase the permeability of cell membrane structure in target region, make the microvessels with the diameter 7 m break down, widen the intercellular space of vascular endothelial cells. The exogenous genes can easily penetrate into the cells and cells through capillary vessels to improve the gene transfection rate and manifestation [12,13]. Cavitation effect can also damage cells, inhibit cell proliferation, and promote tumor cell apoptosis. When ultrasound-targeted microbubble produces strong cavitation effects, it can also damage blood vessel wall, active endogenous or exogenous coagulation, induce large-scale capillary embolism and block nutrient supply to cancerous cells, leading to disappearance of tumor cells [14,15]. Suicide gene therapy has been widely used in liver tumor treatment and showed a good application prospect. Especially the herpes simplex virus thymus kinase/ganciclovir (HSV-TK/GCV) therapy system is most widely applied. HSV-TK is definitely a prodrug enzyme gene which can express and produce TK in the tumor cells, catalyze nucleoside analogue to form mono- phosphate products, and further form Rabbit Polyclonal to PDGFR alpha a triphosphoric acid product under the effect of phosphokinase DMP 777 in the cell. Like a chain terminator, it will interfere with DNA synthesis during cell division, leading to tumor cell death [16,17]. A large number of studies have shown that suicide gene system also has a “bystander effect”. The effect will destroy non-transfected cells with the transfected cells, which overcomes the shortcomings of the low gene transtection rate and greatly enhances the anti-tumor effect of suicide gene therapy [18]. In this study, ultrasound microbubbles wrapped HSV-TK suicide gene experienced targeted launch in mice liver cells, and improved gene transfection effectiveness with the features of ultrasound and microbubbles. In addition, the bystander effect of suicide gene fully played the anti-tumor part. The study offered an efficient, relatively targeted, non-invasive, and physical gene transfection method for HSV-TK/GCV system. == Materials and methods == == Preparation of lipid microbubbles == Dipalmitoyl phosphatidylcholine (DPPC) (sigma, USA), distearoyl phosphatidyl ethanolamine (DSPE) (sigma, USA), diphenyl phosphoryl azide (DPPA) (sigma, USA), glycerol, PBS were mixed relating to a certain proportion and were placed in a 1.5 ml vial, The vials were incubated at 40C for 30 minutes. Each vial was filled with the perfluoropropane gas (C3F8), then the vials were mechanically shaken for 45 mere DMP 777 seconds in a dental care amalgamator (YJT, Shanghai Medical Instrument Co., Ltd.) and quiescence for 5 min. This remedy was diluted by phosphate-bufferedsaline, sterilized by Co60 and stored at 4C;. Then the self-made lipid microbubbles were made. The average diameter was 1.82 0.45 m; the average.
