Objective To investigate whether ultrasound-mediated microbubble destruction can stimulate secretion of vascular endothelial growth factor(VEGF) and angiogenesis in rat myocardium.Methods A total of 30 normal Wistar rats were randomly devided into 3 groups:in group 1,the myocardium was exposed to ultrasound after intravenous injection of microbubbles of 1.0 ml;in group 2,ultrasound was simply used in the myocardium;only was normal saline infused in the control group.Each group was treated 3 times.Thres days after the first experiment,in each group 3 rats were killed and the myocardium were harvested for HE staining to observe the tissue microstructures.The other 27 rats were killed 2 weeks later.Immunohistochemistry stain was used to detect the vascularization.Results There was significant VEGF and CD34 expression in group 1;less in group 2 and little expression in the control.Conclusions Ultrasound-mediated microbubble destruction can facilitate the endogenous secretion of VEGF and accelerate the vascularization in the rat myocardium.

目的研究HIV-1Tat蛋白转导域/质粒DNA/Liposome(TDL)复合物介导增强型绿色荧光蛋白(enhanced green fluorescent protein,pEGFP)在体外培养人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)的转染效率。方法将质粒DNA与Tat肽以不同的电荷比混匀,再加入2μl Lipofectamine2000制成TDL复合物。琼脂糖凝胶电泳分析质粒DNA与Tat肽的结合力;荧光显微镜和流式细胞仪观察TDL复合物与lipofectamine2000介导基因在体外培养人脐静脉内皮细胞中的转染效果;MTT法检测TDL复合物对人脐静脉内皮细胞生长活性的影响。结果TDL复合物组琼脂糖凝胶电泳未发现明显DNA条带。TDL复合物的转染率优于单用Lipofectamine2000(P<0.05)。Tat/DNA电荷比为8∶1时,TDL复合物的转染效率最高。TDL复合物组对细胞活性均无明显影响。结论TDL复合物可明显提高基因在体外培养人脐静脉内皮细胞的转染效率,该方法可作为提高基因转染效率的有效手段之一。

Objective:To construct EGFP and HGF co-expression vector and to detect its expression in cultured eukaryocyte. Methods:A pair of primers of HGF with Sac I and BamH I were designed and constructed,then PCR was performed.The PCR products were inserted into cloning vector pMDlg-T and transferred into JM109.The PCR of colony, restriction analysis,and DNA sequencing were performed to identify the correctness of the recombinant.The HGF gene fragment obtained from pMD18-T/HGF that was digested with Sac I and BamH I,and then inserted into pIRES2-EGFP that was cut with Sac I and BamH I.The recombinant pIRES2-EGFP/HGF was identified with restriction analysis.The expression plasmid pIRES2-EGFP/HGF was transfected into HUVEC mediated by liposome reagent,then the expression of EFGP in cell were ob- served by fluorescence microscopy and HGF protein was detected with Western blotting.Result:The sequence of the cloned DNA fragment was identical to HGF that was reported on Gene bank,and the HGF gene was inserted into eukaryotic expres- sion vector pIRES2-EGFP correctly.The recombinant expression plasmid was successfully transferred into HUVEC observed by fluorescent microscope and effective expression of HGF was also testified by Western blotting.Conclusion:The recombinant eukaryotic co-expression vector of EGFP and HGF was successfully constructed and effectively expressed in HUVEC.

Objective To investigate whether ultrasound-mediated microbubble destruction could effectively enhance the efficiency of liposome delivery plasmid to L 02 cells.Methods The cultured L 02 cells were divided into four groups.The first group was the control group and the second group was given proper dose of liposome with plasmid.Liposome was added with plasmid to the third group and exposed to ultrasound.Liposome with plasmid and microbubbles was applied to the fourth group and exposed to ultrasound.As for the microbubble dosage,the fourth group was devided into 4 subgroups:1.10 μl,2.20 μl,3.30 μl and 4.40 μl groups.One hour after liposome delivered pIRES-EGFP-EHGF to L 02 cells,the fouth group was added 10 μl、20 μl、30 μl or 40 μl microbubbles into each well and exposed to ultrasound.After 24 hours,the pIRES-EGFP-HGF expression in the L 02 cells was detected by fluorescence microscopy,MTT and flow cytometry.Results After exposed to the 1 MHz,0.5 W/cm2 ultrasound for 60 s,30 μl microbubble were added into each well in the 4th group,significantly enhanced efficiency of liposome plasmid transfection was observed.Conclusions In certain condition,the liposome delivered pIRES-EGFP-HGF expression efficiency in L 02 cells was increased with the administration of ultrasound-mediated microbubbles destruction.

Objective Self-made paclitaxel-carrying lipospheres were developed and evaluated as a new ultrasound contrast agent for chemotherapeutic drug delivery.Methods Paclitaxel was added to an aqueous suspension of phospholipids in vial.The headspace of the vials was replace with perfluorobutane gas;the vials were sealed,and they were agitated 30 s on a shaking device.The resulting lipospheres containing paclitaxel were studied for concentration,size,drug entrapment efficiency,drug-loading amounts.Drug release with ultrasound was observed.Results Acoustically active lipospheres containing palitaxel had a mean partical count of approximately(2.3)×10~9～(3.5)×10~9/ml and a mean size of(2.95) μm.The drug entrapment efficiency was more than 90 % and the drug-loading amounts was((26.5)±(0.7))%.The paclitaxel-carrying lipospheres reflect ultrasound as a contrast agent.Fixing amounts of ultrasound energy ruptured the microbubble and released the paclitaxel.The liver imaging of the mice could be enhanced obviously and persistently.Conclusions Liposome microbubbles represent a new class of acoustically active drug delivery vehicles.Future studies will assess efficacy of self-made microbubbles for ultrasound-mediated drug delivery.

超声溶栓的操作参数选择在超声溶栓治疗这一潜在治疗技术中非常重要.聚焦超声作用于体外人全血新鲜血栓,调节其超声参数:作用时间从10 min到60min,强度从7 W/cm2到90W/cm2,频率从2 MHz到4.5 MHz,脉冲作用周期从1:5到1:100或连续波作用,脉冲间隔从100μs到400μs.

Ultrasound-mediated microbubbles destruction as an innovative method for noninvasive delivery of genes and drug are used widely. To choose optimal ultrasound parameters is very important in the treatment of cancer, thrombosis and other diseases by using microbubble deliver gene and drug. This review focuses on the important aspects of ultrasound parameters that are known to influence microbubble-mediated gene and drug delivery.