| Part 1 The Effects of Different Microbubble Concentration and Plasmid Concentration on Transfection Efficiency of pEGFP-N1-VEGFObjective To investigate cell viability of human umbilical vein endothelial cells (HUVEC) and transfection efficiency that would be affected by different microbubble concentration and plasmid concentration of pEGFP-N1-VEGF.Methods HUVEC were cross implanted in the 24 well plate, the control group was exposed to ultrasound only, while the experimental group was added different plasmid concentration(5μl/ml, 10μl/ml, 15μl/ml, 20μl/ml, 25μl/ml, 30μl/ml) and microbubble concentration(0μl/ml, 50μl/ml, 100μl/ml, 150μl/ml, 200μl/ml, 250μl/ml), and then exposed to ultrasound. After 48 hours, cell viability was detected by trypan blue stain, transfection efficiency was observed by fluorescence microscopy and quantitatively detected by flow cytometry, and expression of VEGF was detected by the western-blotting.Result Experimental subgroups had different levels expression of fluorescent protein. When the plasmid concentration was less than or equal with 20μl/ml, the gene transfection efficiency increasd as increasing of plasmid concentration, and the difference was statistically significant (P <0.05). If plasmid concentration was greater than 20μl/ml, the transfection efficiency didn't increase obviously, the difference was not statistically significant (P> 0.05). Cell viability had no significant correlation with plasmid concentration. The wave of transfection efficiency was increased first and then decreased as microbubble concentration increased. when microbbubles concentration was 150μl/ml, the transfection efficiency was the highest. While cell viability was decrsesed as microbubble concentration increased.Conclusions At a certain parameter, the different plasmid concentration and microbubble concentration had different transfection efficiency. Plasmid concentration was 20μl/ml and microbubble concentration was 150μl/ml are ideal conditions for gene transfection.Part 2 The Effects of Different Parameters on Transfection Efficiency of pEGFP-N1-VEGFObjective To investigate cell viability of HUVEC and transfection efficiency of pEGFP-N1-VEGF that would be affected by different ultrasonic parameters. Methods HUVEC were cross implanted in the 24 well plate. The control group only joined with plasmid(20μl) and ultrasound microbubbles(150μl), and the experimental group was exposed to ultrasound. After 48 hours, cell viability was detected by trypan blue stain, transfection rate was observed by fluorescence microscopy and quantitatively detected by flow cytometry, and expression of VEGF was detected by the western-blotting.Result In Control group, there had no expression of fluorescent protein, and western-blotting didn't detecte the expression of VEGF. The experimental groups were seen different levels of fluorescent protein expression. Analysis of four parameters'(frequency, power, duty cycle and the exposure time) main effect showed that the differences were statistically significant (P <0.05). Combinations of any there of the four parameters when there were low frequency, low duty cycle, low power and short exposure time, transfection efficiency increased as the other parameters increased. When the four parameters were high levels, the situations were opposite. In other combinations, transfection efficiency might be rise, fall or increase first and then decrease as the parameters increased. Two of four parameters, three of four parameters, and four parameters had an interaction effect (P <0.05). The cell viability decreased as one parameters increased when the other there ones fixed. It was setted at 1MHZ, 1.5 W/cm2, 20% duty cycle, 120s that the transfection efficiency was highest. It was an ideal optimization transfection parameters.Conclusions Optimization of transfection parameters on cell death and higher transfection efficiency is expected to become an effective and safe gene transfection strategy. |
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