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ObjectivesTO construct ultrasound microbubble carrying pDsRed2-N1-CD/UPRT fusion suicide gene,To study the lethal effect of ultrasound microbubble combined with pDsRed2-N1-CD/UPRT fusion suicide gene for lung cancer in vitro.MethodsCytosine Deaminase(CD)and uracil phosphoribosyltransferase (UPRT) genes of E.coli were amplified by PCR and cloned into vector pDsRed2-N1.The constructed recombinant plasmid pDsRed2-N1-CD/UPRT was transformed to E.coli BL21(DE21),and the positive recombinants were screened,from which plasmid was extracted for restriction analysis.The recombinant plasmid was fused with ultrasound microbubble,and the mixture was tranfected to A549 cells to observe the direct killing and side effects of CD/UPRT gene.The effects of ultrasound at various strengths on the plasmid structre in mixture of ultrasound microbubble and recombinant plasmid,morphology of transfected cells and tranfection efficacy were detemined during different time.ResultsRestriction analysis proved that recombinant plasmid pDsRed2-N1-CD/UPRT was constructed correctly,The distribution of recombinant plasmid in ultrasound microbubble was observed by fluorescent microscopy.The inhibitory rate to growth of A549 cells which transfected ultrasound microbubble combined with recombinant plasmid pDsRed2-N1-CD/UPRT increased with the increasing concentration of 5-FU,which reached 58%,Compared with the control group (blank group group A549 cells, A549 cells + plasmid group, A549 cells + microbubble group), there were significantly different (P <0.05).However,at the same 5-FU dosages,the inhibitory rate with the increasing proportion of transfected cells carrying microbubbles combined with recombinant plasmid pDsRed2-N1-CD/UPRT,which reached 78%, Compared with the control group (without 5-FU treatment of A549 cells, A549 cells + plasmid group, A549 cells + microbubble group), there were significantly different (P <0.05). To observe biological behaviors of the plasmid in different energy ultrasonic,found the electrophoretic band of plasmid pDsRed2-N1-CD/UPRT which showed no significant change after treatment by ultrasound with low energy,indicating no significant effect on plasmid structure.The ultrasound with low energy showed no significant effect on the morphology of transfected cells,and the ultrasound-mediated gene transfection efficacy reached 30% at most. Increased ultrasonic energy and time, we found transfection efficiency of pDsRed2-N1-CD/UPRT plasmid significantly decreased,and signs of apoptosis.ConclusionUltrasound microbubble enhanced the directly killing and side effects of CD/UPRT gene on tumor cells,thereby increased lethal effect in tumor cells.Under treatment of low-energy ultrasound,the ultrasound microbubble with low energy increased the tranfection efficacy of cells with CD/UPRT gene while showed no damage to cell and DNA of target gene.Ultrasound treatment for peripheral lung cancer, which will improve the theoretical basis for targeted therapy.
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