The Study Of Ultrasound Mediated Microbubble Destruction To Enhance Liposome Gene Transfection In Liver Cell

PARTⅠOPTIMIZATION OF PARAMETERS IN ULTRASOUND MEDIATED MICROBUBBLE DESTRUCTION ENHANCE GENE DELIVERY IN L02 CELLObective: To observe the growth condition of human liver cell line L02 treated by ultrasound mediated microbubble destruction and to optimize the parameters of ultrasound mediated microbubble destruction for gene transfection .Method: The growth condition of L02 cell treated by ultrasound, microbubble and ultrasound mediated microbubble destruction was detected by MTT. The gene transfection in L02 cell treated by ultrasound mediated microbubble destruction was detected by fluorescence microscopy and flow cytometry . The ultrasound parameter and microbubble dosage was optimized for ultrasound mediated microbubble destruction for gene transfection . Results: MTT resultes showed the survival rate of L02 cell is 92.45%, 85.78%, 31.65% ,when ultrasound frequency was 1MHz ,intensity was 0.5 W/cm~2 ,duration was 30s, 60s, 90s . Different dose of microbubble was added into L02 cell . 24 hours later there was no significant difference in the survival rates between the experiment groups and the control group. Ultrasound mediated microbubble(40μl) destruction could reduce the survival rate of L02 cell significantly,while ultrasound mediated microbubble (10μl,20μl,30μl) destruction doesn't have much impact on L02 cell. The fluorescence microscopy found that the green fluorescence can be seen both in ultrasound mediated microbubble destruction groups and liposome group. Flow cytometry result showed the transfection efficiency of liposome group and ultrasound mediated microbubble destruction 10μl group,20μl group,30μl group, 40μl group was 18.30%,10.14%,14.65%,18.54%,10.06% respectively.Conclusion:Ultrasound mediated microbubble destruction enhance the transfection efficiency of EGFP in L02 cell and has no significant impact on it's growth condition ,when ultrasound frequency was 1MHz, intensity was 0.5 W/cm~2 ,duration was 60s and microbubble concentration was 1.8×10~7个/ml. PARTⅡULTRASOUND-MEDIATED MICROBUBBLE DESTRUCTION ENHANCE THE EFFECIENCY OF LIPOSOME DELIVERY PLASMID TO L02 CELLSObjective: To detect the transfection efficiency of pIRES-EGFP/HGF in L02 cell treatd by ultrasound mediated microbubble destruction combined with liposome.Method: L02 cells was divided into 5 groups ,①control group,②liposome group,③ultrasound irradiation liposome group,④ultrasound mediated microbubble destruction group,⑤ultrasound mediated microbubble destruction + liposome group.The transfection efficieny of pIRES-EGFP/HGF in L02 cells was observed and detected by fluorescence microscope and flow cytometry. L02 cell's growth rate and HGF contents in clear supernatant liquid of L02 cell was detected by MTT and ELISA.Results: Flow cytometry results showed the transfection efficiency of liposome group ,ultrasound irradiation group, ultrasound mediated microbubble destruction group and ultrasound mediated microbubble destruction + liposome group were 18.32%,18.46%,18.47%l23.17%.The control group wasn't seen gene transfection .ELISA results showed the HGF contents in L02 cell's clear supernatant liquid of ultrasound mediated microbubble destruction + liposome is the highest .MTT indicated the growth rate of liposome group ,ultrasound irradiation group, ultrasound mediated microbubble destruction group and ultrasound mediated microbubble destruction + liposome group were 100%,117.79%,119.52%,118.44%,123.21% respectively.Conclusion: Ultrasound mediated microbubble destruction can enhanced liposome deliver pIRES-EGFP/HGF to L02 cell significantly.The transfection efficiency is much higher than ultrasound mediated microbubble group ,ultrasound irradiated liposome group and liposome group .