超声介导载基因微泡靶向传输血管内皮生长因子促心肌血管新生

Proangiogenesis of infracted myocardium induced by human vascular endothelial growth factor (VEGF_(165)) directed by the targeted delivery system of ultrasound mediated bearing-gene microbubbles' cavitation

目的 探讨超声介导载血管内皮生长因子 16 5 (VEGF165)基因靶向微泡促梗死心肌血管新生的可行性。方法 构建真核表达质粒 pcDNA3 1-/VEGFcDNA165,将其包载于脂质泡中 ,超声辐射下向鼠心肌传输。采用RT PCR、WesternBlot检测mRNA、蛋白表达 ,观察微血管密度评价血管新生效果。结果 (1)成功构建VEGF165基因 ;(2 )脂质体微泡可包载VEGF165基因 ;(3)超声介导辐射下向心肌靶向传输VEGF165基因 ,实验组基因表达及血管密度高于空白对照组 ,但低于VEGF165基因心肌直接注射。结论 具有心肌显影功能的脂质体载VEGF165基因微泡在超声辐射下可向大鼠梗死心肌靶向传输 ,并产生促血管新生效应。

Objective To explore the feasibility of directed delivering human vascular endothelial growth factor (VEGF_ 165 ) to induce therapeutic angiogenesis in rat′s infracted myocardium zone with ultrasound mediated microbubbles′ destruction which bear angiogenic growth factor gene. Methods VEGF_ 165 complementary DNA was obtained by reverse transcriptase polymerase chain reaction(RT-PCR) from mRNA extractions of human lung tissue of a four-month old normal aborted fetus, and then it was cloned into a high expressing efficiency eukaryotic plasmid pcDNA3.1 - after sequence analyses and double endonuclease cutting was accomplished. Partial physical and chemical property of prepared liposome microbubble and its functions including targeted delivery of VEGF_ 165 to infracted myocardium in rats were investigated. Two weeks after gene delivery, the hearts were harvested. RT-PCR and Western Blot analyses were carried out to evaluated mRNA and protein expression of VEGF_ 165 respectively. Micro vessels density(MVD) counting of infracted myocardium in rats observed by CD_ 34 immunochemical staining was conducted to value the pro-angiogenesis effect of VEGF_ 165 delivered by ultrasound mediated cavitation of liposome microbubble bearing VEGF_ 165 gene. Results (1)Recombinated eukaryotic expression plasmid DNA encoding pcDNA3.1 -/VEGF_ 165 were successfully constructed and confirmed by sequence analyzing and endonuclease cutting. (2) DNA material could be wrapped into or adhere to the prepared liposome microbubble verified by electron microscope observing. Microbubble parameter quantification showed that its diameter was less than five micron and it could easily pass dog pulmonary circulation and cause myocardium developing continuously for almost seven minutes during myocardial contrast echocardiography after intravenous bolus injection in a canine model. (3) After delivering by ultrasound mediated microbubble cavitation, the expression of VEGF_ 165 mRNA and its protein and MVD counting were just inferior to direct VEGF_ 165 gene myocardium injection. Conclusion The explored liposome microbubble which had the potential function as myocardial contrast agent, could bear and deliver genes such as VEGF_ 165 into infracted myocardium of rats and produce angiogenesis effect.