| Objective To evaluate ultrasound and microbubble contrast agent enhance the enhanced green fluorescent protein reporter gene plasmid (pEGFP) in rat vascular smooth muscle cells (VSMC) transfection efficiency, and whether it will lead to changes in cell activity.Methods Experiment was divided into A,B,C,D four groups, which were the control group,simple plasmid immersion group, plasmid+ ultrasonic irradiation group, plasmid+ ultrasonic irradiation+ microbubble contrast agent group. The rat VSMCs was exposure by the diagnostic ultrasonography for 10min in vitro add or without ultrasound microbubble contrast agent, and the ultrasonic probe frequency was 10 MHz, mechanical index was 1.9, pEGFP concentration in the solution was 5μg/m.l. The cells was observed by Trypan blue staining to detect cell activity (survival rate) just after exposure, Transfection efficiency of pEGFP was observed by the fluorescent microscopy after 48h of the exposure.Results The cell survival rates of A, B, C, D four groups were (97.43±2.63)%, (97.52±1.64)%, (96.83±±2.31)% and (95.21±1.36)%, there were no significant difference between the groups, Transfection efficiency of pEGFP in A, B, C, D four groups were 0.12±0.42)%,(1.31±0.71)%,(4.45±3.13)% and(21.48±2.16)%, there was significant difference between D group with the others.Conclusions The ultrasound and microbubble contrast agent can significantly improve the pEGFP transfection efficiency in VSMC, whereas no influence on the activity of cells.Objective To investigate the feasibility and efficacy of ultrasound and microbubble contrast agent mediated endothelial nitric oxide synthase gene transfer in rat vascular smooth muscle cells (VSMC).Methods Experiment was divided into A,B,C,D,E five groups, which were the control group, simple plasmid immersion group, plasmid+ microbubble contrast agent group, plasmid+ ultrasonic irradiation group, plasmid+ ultrasonic irradiation+ microbubble contrast agent group. The rat VSMCs was exposure by the diagnostic ultrasonography for 10min in vitro add or without ultrasound microbubble contrast agent, and the ultrasonic probe frequency was 10 MHz, mechanical index was 1.9, the plasmid of pcDNA3.1-eNOS concentration in the solution was 5μg/ml.RT-PCR,Western blotting Were used to detecte the eNOSmRNA and protein express in VSMC after 48h of the exposure.Results The expression of eNOSmRNA in plasmid+ ultrasonic irradiation+ microbubble contrast agent group was most significant, IOD ratio was(91.11±3.41)%, simple plasmid immersion group, plasmid+ microbubble contrast agent group and plasmid+ ultrasonic irradiation group also has a small amount of expression, IOD ratios were (26.10±1.32)%%, (31.42±2.43)%, (35.05±2.25)% respectively, P<0.05compared with the E group.Western blot analysis of eNOS protein expression, and control group had very little expression, simple plasmid immersion group, plasmid+ microbubble contrast agent group and plasmid + ultrasonic irradiation group also has a small amount of expression, IOD ratios were (22.12±1.33)%, (25.42±2.41)%, (33.11±3.11)%, plasmid+ ultrasonic irradiation+ microbubble contrast agent group expressed the most obvious, IOD ratio was (84.22±9.22)%, P<0.05 compared with the others.Conclusions The ultrasound and microbubble contrast agent can significantly improve the eNOS transfection efficiency in VSMC, and Increased nitric oxide synthase in VSMC.Objective To investigate the feasibility and efficacy of ultrasound and microbubble contrast agent improve gene transfection into rat venous bypass grafting.Methods Sprague-Dawley rats underwent interposition bypass grafting of the common carotid artery via the ipsilateral external jugular vein. Before anastomosis, the vein segment was put into the solutions with microbubbles which were adhered with plasmid enhanced green fluorescent protein (pEGFP), Pulsed Doppler ultrasound was used for 10 mintues. The grafts were harvested in 2d after surgery respectively. The expression of pEGFP in vascular smooth muscle cells was examined by fluorescence microscopy, and meanwhile, the vascular was observed with hematoxylin-eosin staining. Results The green fluorescence was visualized more in the group of adhesion pEGFP Microbubble contrast agent plus ultrasound, which the fluorescent intensity was higher than that in group ultrasound exposed only and that in group dipping with pEGFP only. There was no obvious tissue necrosis in every group.Conclusions Microbubble contrast agent plus ultrasound exposure can significantly improve the transfection efficiency of pEGFP in rat vein graft.Objective To investigate the feasibility and efficacy of ultrasound and microbubble contrast agent mediated nitric oxide synthase gene transfer prevents restenosis of vein graf.Methods Sprague-Dawley rats underwent interposition bypass grafting of the common carotid artery via the ipsilateral external jugular vein. Before anastomosis, the vein segment was put into the solutions with microbubbles which were adhered with the plasmid of pcDNA3.1-eNOS, Pulsed Doppler ultrasound was used for 10 mintues. The grafts were harvested in 28d after surgery respectively. the vascular was observed with hematoxylin-eosin staining and Immunohistochemical staining and Western blot analysis respectively to evaluate the extend of re-stricture of the vessels.Results The expression of eNOS gene in the vein grafts in the Plasmid+ microbubble contrast agents+ irradiation group was stronger than that in model control group, pure plasmid immersion group, Plasmid+ ultrasonic irradiation group and the plasmid+ microbubble contrast agent group, significantly decreased intimal hyperplasia, the degree of restenosis was significantly lower than other control group also.Conclusions Ultrasound and microbubble contrast agent mediated nitric oxide synthase gene transfer can inhibit the vein graft neointimal hyperplasia, which can provide a new idea for the prevention of graft restenosis in gene therapy.
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