Abstract
Gene therapies have been applied to the treatment of cardiovascular disease, but their use is limited by the need to deliver them to the right target. We have employed targeted contrast ultrasound-mediated gene transfection (TCUMGT) via ultrasound-targeted microbubble destruction (UTMD) to transfer therapeutic genes to specific anatomic and pathological targets. Phospholipid microbubbles (MBs) with pcDNA3.1-human vascular endothelial growth factor 165 (pcDNA3.1-hVEGF165) plasmids targeted to P-selectin (MB+P+VEGFp) were created by conjugating monoclonal antibodies against P-selectin to the lipid shell. These microbubbles were divided into four groups: microbubble only (MB), microbubble+P-selectin (MB+P), microbubble+pcDNA3.1-hVEGF165 plasmid (MB+VEGFp), and microbubble+ P-selectin+pcDNA3.1-hVEGF165 plasmid (MB+P+VEGFp). The reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) results showed that the VEGF gene was successfully transfected by TCUMGT and the efficiency is increased with P-selectin targeting moiety. UTMD-mediated delivery of VEGF increased myocardial vascular density and improved cardiac function, and MB+P+VEGFp delivery showed greater improvement than MB+VEGFp. This study drew support from TCUGMT technology and took advantage of targeted ultrasound contrast agent to identify ischemic myocardium, release pcDNA3.1-hVEGF165 recombinant plasmid, and improve the myocardial microenvironment, so promoting the restoration of myocardial function.
摘要
目的
构建一种靶向阳离子微泡,探讨其提高超声靶向 击碎微泡技术(TCUMGT)介导的体内基因转染 效率及治疗效果。
创新点
提出运用TCUMGT 介导的基因转染技术,利用 微泡的携基因和靶向定位释放双项功能来上调 缺血区的人血管内皮生长因子165(hVEGF165) 的表达水平,发挥其成血管作用,从而改变缺血 心肌的存活性。
方法
通过聚乙二醇40 硬脂酸酯、二硬脂酰基磷脂酰 乙醇胺-聚乙二醇2000、pcDNA3.1-hVEGF165 和 抗P-选择素单克隆抗体等制备P-选择素靶向阳 离子微泡。微泡分成四组:(1)仅微泡(MB); (2)微泡+P-选择素(MB+P);(3)微泡+pcDNA3.1-hVEGF165质粒(MB+VEGFp);(4)微泡+P-选 择素+pcDNA3.1-hVEGF165质粒(MB+P+VEGFp)。 逆转录聚合酶链反应(RT-PCR)和酶联免疫吸 附试验(ELISA)结果显示:TCUMGT 成功转 染hVEGF165 基因,并且通过P-选择素为靶点可 以提高转染效率。另外与其他组相比,MB+P+ VEGFp 组的心肌血管密度增加和心功改善最为 明显。
结论
本研究表明,通过TCUMGT 技术,靶向超声微 泡可以有效识别缺血心肌,释放pcDNA3.1-hVEGF165 重组质粒,提高心肌微环境,促进心 肌功能的恢复。
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Acknowledgements
Special thanks go to Prof. You-bin DENG from Department of Ultrasonography, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, for his comments and suggestions in the writing process of the article.
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Project supported by the Natural Science Foundation of Zhejiang Province (No. LY14H180003), the National Natural Science Foundation of China (No. 81301231), and the General Research Project of Zhejiang Provincial Department of Education (No. Y201636244), China
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Shentu, Wh., Yan, Cx., Liu, Cm. et al. Use of cationic microbubbles targeted to P-selectin to improve ultrasound-mediated gene transfection of hVEGF165 to the ischemic myocardium. J. Zhejiang Univ. Sci. B 19, 699–707 (2018). https://doi.org/10.1631/jzus.B1700298
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DOI: https://doi.org/10.1631/jzus.B1700298
Key words
- Vascular endothelial growth factor (VEGF)
- P-selectin
- Targeted contrast ultrasound-mediated gene transfection
- Heart function