超声破裂载基因微泡增强心肌细胞报告基因的转染与表达

ENHANCED REPORTER GENE TRANSFER AND EXPRESSION IN CARDIAC MYOCYTES MEDIATED BY ULTRASONIC DESTRUCTION OF THE MICROBUBBLES

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。

Aim： To determine if the diagnostic ultrasound and self-made microbubbles could be used to increase gene transfection and expression in cardiac myocytes by means of the ultrasound-mediated microbubbles destruction. Methods： The perfluoropropane-exposed sonicated dextrose albumin（PESDA） microbubbles were made and mixed with indicated volume reporter gene encoding β-galactosidase prior to gene transfection. Gene transfeetion into the cultured cardiac myocytes was performed by exposure to the various intense diagnostic ultrasound（ 1.3 MHz） in the presence of the gene-attached microbubbles. The calcium phosphate precipitation gene transfection was carried out alone or in combination with ultrasound-mediated destruction microbubbles. The cells were harvested 48 h after transfection and β-galactosidase expression was detected by in situ staining and quantitive assay. Results： Cardiac myocytes exposed to ultrasound with PESDA induced significantly increase in gene expression（60-fold compared with naked plasmids transfection, P 〈 0.01）. Moreover, it was found that the reporter gene expression not only related with ultrasound intension but also with the microbubbles concentration. In combination with calcium phosphate precipitation gene transfection, ultrasound-mediated destruction microbubbles resulted in more intense gene expression even 6 hours after calcium phosphate precipitation gene transfection. Conclusion： The ultrasonic destruction of gene-loaded microbubble is a highly effective gene transfer method, and it not only acts on the gene entry into cells, but also on the intracellular exogenous DNA expression.