Ultrasound And Microbubble: Their Functions In Gene Transfection In Vitro And In Vivo

Part 1 Ultrasonic Exposure-time and Microbubble-dosage: Their Effect on Gene Transfection in HepG2 Cells in VitroObjective To investigate the relationship of gene transfection efficiency with different ultrasound exposure time and dose of microbubble(MB) mediated gene transfection,and to find the appropriate ultrasound parameters for gene transfection.Methods Plasmid encoding enhanced green fluorescent protein(pEGFP)was chosen as the report gene and HepG2 cells was chosen as the research object. The HepG2 cells plus pEGFP and different dose of microbubble were exposed to ultrasound (1MHz, 0.5W/cm2) for varying time. After twenty-four hours, the expression of EGFP in the cells was observed by fluorescence microscope, the transfection efficiency was assessed by FACS and the cell viability was detected by trypan blue exclusion.Results The expression of EGFP in every experimental group was different, and the approving transfection efficiency was got by ultrasound exposed for 20s meanwhile the dose of microbubble was 60μl. Conclusions With fixed ultrasound frequency and power, different transfection efficiencies were got when the exposure time and the doses of microbubble were different. The appropriate parameter was 20s, 60μl. Those upon can supply some informations for further study. Part 2 Microbubbles and Pluronic P85:The Preliminary Experiment Research of Their Functions in Gene Transfected by Ultrasound in vivoObjective To investigate whether local ultrasound(US) irradiation could enhance the local gene transfection and expression after intravenous injection of pluronic P85 and plasmid which adhered on the microbubble(MB) in vivo.Methods Plasmid encoding enhanced green fluorescent protein(pEGFP)was chosen as the report gene. The model of hepatic carcinoma in nude mice was established by injected HepG2 cells subcutaneously. pEGFP was injected into the tail vein with transfection solution. Experimental Balb/c-nu mice were randomly divided into three groups, pEGFP+US group (the first group), pEGFP+MB+US group (the second group) and pEGFP+MB+P85+US group (the third group). Pulsed Doppler ultrasound was used (1MHz,0.5W/cm2) for 2 minutes. After seven days, the expression of EGFP in hepatocarcinoma cell was examined by in-vivo fluorescence, then the mice were killed and the subcutaneous tumors were removed and did rapid frozen section. The transfection efficacy was assessed by counting the number of EGFP-positive cells under fluorescence microscopy, and the other sets of sections were stained with haematoxylin and eosin to show histopathology changes of tumor in animals of different groups.Results There were many green fluorescence-expressing cells in the second and the third group, and those were fewer in the first group. In the third group, the level of expression of EGFP was significantly higher than that in the first and the second group (P<0.05), and the level of the second group was significantly higher than that in the first group too (P<0.05). In all the groups, there were no significant differences in tissue damage through HE. Conclusions Local ultrasound irradiation after intravenous injection of Pluronic P85 and plasmid which adhered on the microbubble could significantly enhance the local gene transfection and expression; it may have a certain synergistic effect for gene transfection between microbubble and Pluronic P85, and there was no damage to the tissues. This method may provide a safe gene delivery technique in vivo, and it deserves further attention and investigation.