新型载基因微泡的制备及其在心肌细胞报告基因转染中的应用

Synthesis of New Gene-loaded Microbubbles Serve as Gene Delivery Vehicle Applied in Reporter Gene Transfer into Cardiac Myocytes

为了提高基因黏附微泡的稳定性和基因携带容量,采用改良超声声振法将质粒-多聚乙酰亚胺(PE I)复合物整合至微泡包膜上而制备出新型载基因微泡。电泳分析及细菌转化实验表明PE I能降低超声声振对质粒结构及功能的破坏。新型载基因微泡具有良好的声学及血液流变学性能,其基因携带量明显高于基因黏附微泡。分别采用超声破裂新型载基因微泡及基因黏附微泡介导心肌细胞β-半乳糖酶基因转染。结果表明,超声破裂载基因微泡能增强裸质粒转染效率达107倍,其基因表达水平为超声破裂基因黏附微泡组的6.85倍。提示经改良法制备的新型载基因微泡是一种安全高效的基因转运载体,超声破裂载基因微泡能明显增强心肌细胞的基因转染效率。

To improve the stability and gene-carried capability of gene-attached microbubbles, the method for manufacture of albumin microbubbles was modified and new gene-loaded microbubbles were synthesized by incorporated gene-PEI complex into the shell of microbubbles. Agarose gel electrophoresis and bacteria transformation showed that PEI had the ability to provide the protection of plasmid DNA from ultrasonic degradation. The new gene-loaded microbubbles exhibited excellent acoustical and hemorheological properties. Moreover, they could carry more plasmid DNA than gene-attached microbubbles. β-galactosidase plasmid tansfection into cardiac myocytes was performed by using ultrasound targeted destruction of new gene-loaded microbubbles or gene-attached microbubbles. Gene expression in cardiac myocytes was detected by β-galactosidase in situ staining and quantltlve assay. It was shown that β-galactosidase activity in cardiac myocytes was enhanced 107-fold by ultrasonic destruction of gene-loaded microbubbles compared with naked plasmid transfection and new gene-loaded microbubbles resulted in 6.85-fold increase in β-galactosidase activity compared with optimal transfectlon mediated by gene-attached mlcrobubbles. These results suggested that ultrasonic destruction of the gene-loaded microbubbles can enhance the cardiac myocytes exogenous gene transfer efficiency significantly and new gene-loaded microbubble is an efficient and safe gene delivery vehicle.