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目的:通过超声破裂载基因微泡介导报告基因心肌细胞转染,探讨其能否增强心肌细胞外源基因转染与表达。方法:以-βgalactosidase质粒为报告基因,将其与自制氟碳气体微泡粘附,制备载基因微泡。利用诊断性超声破裂微泡进行体外心肌细胞基因转染;以磷酸钙共沉淀转染为阳性对照并将其以不同方式与超声破裂微泡技术联合应用,以期进一步增强基因转染效果。分别采用原位染色及酶学定量检测-βgalactosidase表达水平,同时进行细胞活性检测。结果:超声破裂载基因氟碳气体微泡(PESDA)转染组心肌细胞-βgalactosidase表达水平可达单纯质粒转染组60倍(P<0.01)。磷酸钙共沉淀转染3.67倍(P<0.01)超声强度、微泡浓度对超声破裂介导基因转染效果有明显影响。超声破裂微泡技术与磷酸钙共沉淀联合应用可进一步提高报告基因的表达(P<0.05),即使在磷酸钙转染后6 h,超声破裂微泡仍能明显增强报告的基因的表达(P<0.05)。结论:超声破裂微泡技术是一种高效基因转染方法,其不但能增加DNA转染,而且增强入胞后基因的表达。超声破裂微泡与其它基因转染技术联合应用能进一步增加基因转染效率。
OBJECTIVE: To transduce reporter gene cardiomyocytes mediated by ultrasound-mediated gene-loaded microbubbles to investigate whether it can enhance the transfection and expression of foreign genes in cardiomyocytes. Methods: The β-galactosidase plasmid was used as a reporter gene to adhere to self-made fluorocarbon gas microbubbles to prepare gene-loaded microbubbles. In vitro cardiomyocyte gene transfection with diagnostic ultrasound rupture microbubbles; calcium phosphate coprecipitation transfection as a positive control and its use in combination with ultrasonic rupture microbubbles in different ways, in order to further enhance the gene transfection. The expression of β-galactosidase was detected by in situ staining and enzymology respectively, and the cell viability was also detected. RESULTS: The expression level of β-galactosidase in cardiomyocytes transfected with fluorogenic carbon gas microbubble (PESDA) was up to 60 times higher than that in the plasmid transfected group (P <0.01). Calcium phosphate co-precipitation transfection of 3.67 times (P <0.01) ultrasound intensity, microbubble concentration ultrasound transfection-mediated gene transfection have a significant effect. Ultrasound ruptured microbubble technique combined with calcium phosphate co-precipitation could further increase the expression of reporter gene (P <0.05). Ultrasound ruptured microbubbles could enhance the expression of the reported gene even after 6 h transfection of calcium phosphate (P <0.05). Conclusion: Ultrasonic rupture microbubble technique is an efficient method of gene transfection, which not only increases DNA transfection, but also enhances gene expression after transfection. Ultrasound rupture microbubbles combined with other gene transfection techniques can further increase the gene transfection efficiency.