Experiment Preparation Of HSV-TK Gene Targeting Ultrasound Microbubble And Targeting In Vitro

part I targeting lipid ultrasound microbubble preparationObjective: Preparation of a lipid ultrasound microbubble as carrier,make its particle size of nanoscale, and implement it with liver cancer cellspecificity antibody coupling.Methods:1. DPPA, Bio-DSPE according to certain mole ratio ofdissolved in organic solvents, After being volatile organic solution, form ofwhite powder material, take out a certain amount of material, the glycerin,PBS buffer being added,45℃water bath then injection of C3F8gas,ultrasonic microvesicles can be prepared by the mechanical shock methods.ultrasound microbubble been observationed under light microscopy and itsdiameter size and surface Zata potential been measuremented.2.The antibodies and ultrasound microbubble been linked by biotin-avidin system, microbubble form been observed under fluorescencemicroscope after take in FICT labeled second antibodies. Flow cytometryinstrument detection the ratio of targeted ultrasound microbubble.Results:1.The ultrasound microbubble into a round shape, the size ismore uniform, verage particle size of about520nm, the zata-22mv.2.observation of targeted ultrasound microbubble under fluorescence microscope, under blue light excitation wave will observation microbubblecenter for black, surrounded by a ring of green fluorescence, Show thatGPC3monoclonal antibody has specific binding to the microbubble surface.Flow cytometry instrument detection the ratio of targeted ultrasoundmicrobubble is85Percentage.Conclusions:1.The ultrasound microbubble size is uniform, goodstability, the particle size of nanoscale, the average particle size of less than700nm, can through interstitial tumor vascular endothelial cells.2. theultrasound microbubble coupling with GPC3monoclonal antibody by biotin-avidin system, provides the basis for target identification and follow-upstudy. Part II target experimental of liver cancer cells in vitroObjective: The connection with GPC3monoclonal antibody ofultrasound microbubble,Realize specificity combined with humanhepatocellular carcinoma HepG2cells.Methods:1. The cultivation of human hepatocellular carcinoma HepG2cells and human liver L02cell, from Cell immunohistochemical experimentto confirmed liver cancer has a expression of GPC3antigen on the cellmembrane surface.2. HepG2cells, L02cell and human breast cancer cell MCF–7via DiIdyeing, take in targeted ultrasound microbubble which conjoin FITC markantibodies, detection of the target by laser confocal waiting for its fullyintegrated. Results:1. HepG2cells can express GPC3antigen on cell membranesurface but L02cell can not.2.Targeted ultrasound microbubble canspecificity conjoin HepG2cell membrane surface, L02cell and MCF–7cellcan not.Conclusions: Targeted ultrasound microbubble specific identificationof liver cancer cells in vitro can be realized in the study. Part III Load HSV-TK gene targeted ultrasoundmicrobubble preparationObjective: Preparation of a kind of ultrasound microbubble which canalso link GPC3monoclonal antibody and HSV-TK gene.Methods:1.In order to realize HSV-TK gene conjoin to microbubble,we will take the mixture of Poly-l-Lysine and HSV-TKgene in microbubbleobserved microbubble form under laser confocal after join PI which canmark plasmid.2. On the basis of targeted ultrasound microbubble link HSV-TKsuicide gene, Preparation of Load HSV-TK gene targeted ultrasoundmicrobubble and observed under laser confocal.Results:1. After PI mark Load HSV-TK gene ultrasound microbubble,microbubble center is black, with a circle around the red fluorescence beenobserved under laser confocal green light excitation, which show that HSV-TK gene specificity combined with micro bubble surface.2. Load HSV-TKgene argeted ultrasound microbubble under blue light excitation wavemicrobubble surrounded by a ring of green fluorescence, under green light excitation wave microbubble with a circle around the red fluorescen. greenand red fluorescence can complete overlap, form yellow fluorescence underconfocal laser processing, so it is prove microbubble surface links withGPC3antibodies and HSV TK suicide gene.Conclusions: Successful preparation of Load HSV-TK gene argetedultrasound microbubble, it is establish the theoretical basis for genetransfection in vitro and in vivo tests. Key words: HSV-TK, GPC3, targeted ultrasound microbubble...