超声介导微泡靶向传输基因促血管新生的实验研究

Experimental research on therapeutic angiogenesis induced by human vascular endothelial growth factor directed by targeted delivery system of ultrasound mediated cavitation of liposome microbubbles

目的探讨超声介导微泡靶向传输血管内皮生长因子VEGF165促心肌血管新生即心肌“分子搭桥”的新途径。方法利用分子生物学方法构建人血管内皮生长因子真核表达质粒pcDNA3.1-/VEGF165;制备载VEGF165基因脂质体微泡,分析其理化性质和功能,在超声场利用超声辐射向大鼠梗死心肌靶向传输VEGF165;2周后取材,逆转录聚合酶链式反应(RT PCR)评价大鼠心肌中人源VEGF165mRNA表达,蛋白印迹杂交(WesternBlot)观察大鼠心肌VEGF165的蛋白表达,CD34免疫组化微血管密度(MVD)计数半定量法评价超声介导微泡靶向传输VEGF165促梗死心肌血管新生效果。结果①成功构建、克隆血管内皮生长基因VEGF165重组真核表达质粒pcDNA3.1-/VEGF165,测序分析、酶切鉴定准确无误;②研制的脂质体微泡超微结构显示可粘附或包载DNA,粒度分析显示平均粒径<5μm;③在超声介导下该脂质体载基因微泡可靶向传输VEGF165至大鼠梗死心肌,超声介导靶向传输组VEGF165的mRNA、蛋白表达及促血管新生作用(MVD表示)仅次于VEGF165基因心肌注射组。结论超声介导微泡可向大鼠心肌靶向传输VEGF165基因并产生促血管新生效应。

Objective To explore a novel approach for directing human vascular endothelial growth factor (VEGF165) to induce therapeutic angiogenesis in rat′s infarcted myocardium zone. Methods VEGF165 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 to high efficiency eukaryotic expressing plasmid pcDNA(3.1)^- 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 VEGF165 to infarcted myocardium in rats were investigated. Two weeks after gene delivery,RT-PCR and western blot analyses were conducted to evaluate mRNA and protein expression of VEGF165 respectively.Microvessels density(MVD) counting of infracted myocardium,observed by CD34 immunochemical staining,was performed to value the proangiogenesis effect of VEGF165 delivered by ultrasound mediated cavitation of liposome microbubble bearing VEGF165 gene. Results ①Recombinated eukaryotic expression plasmid DNA encoding pcDNA(3.1)^-/VEGF165 was successfully constructed confirmed by sequence analyzing and endonuclease cutting.②Microbubble parameter quantification shows its diameter was less than (5 μm) by COULT counter′s analysis and electron microscope observing.③After delivering by ultrasound mediated microbubble cavitation,the expression of VEGF165 at mRNA and protein level and MVD counting were just inferior to VEGF165 gene myocardium injection. Conclusions The explored liposome microbubble cavitated by ultrasound radiation could deliver genes such as VEGF165 to infracted myocardium of rats and produce angiogenesis effect.