超声微泡介导pEGFP-N1质粒转染人牙周膜成纤维细胞的实验研究

目的 探讨超声微泡造影剂在一定能量的超声波辐照下,介导质粒pEGFP-N1转染人牙周膜成纤维细胞(HPDLFs)的效率及安全性.方法 体外原代培养HPDLFs,以EGFP基因为报告基因,脂质微泡造影剂为载体,用超声辐照介导质粒pEGFP-N1转染HPDLFs.实验组超声+微泡+质粒组根据转染条件不同分成不同亚组,对照组为质粒组、微泡+质粒组、超声+质粒组和脂质体+质粒组.转染48 h后在倒置荧光显微镜下观察绿色荧光蛋白GFP表达.同时用MTT法检测HPDLFs的活力.结果 超声微泡介导的质粒对HPDLFs的转染效率与脂质体介导的质粒转染效率相似,明显高于其他对照组.超声+微泡+质粒组中HPDLFs的活力明显高于脂质体+质粒组.结论 在一定条件下,超声微泡能安全、有效地介导外源基因的转染与表达.     

超声微泡造影剂介导小鼠骨骼肌基因转染实验研究

目的探讨微泡造影剂在超声作用下是否可增加小鼠骨骼肌基因转染效率.方法 40只昆明小鼠随机分为4组,每组10只,第一组:在胫前肌注射造影剂与绿色荧光蛋白(GFP)质粒的混合溶液;第二组:注射与第一组相同的混合溶液后立即加超声辐照;第三组:注射GFP;第四组:在注射GFP后立即用超声辐照.7天后取小鼠胫前肌观察绿色荧光蛋白的表达情况.结果第一组与第二组有较多GFP表达,部分肌纤维绿色荧光较明亮,部分较暗淡;第三组和第四组GFP表达量较少.第一组与其余各组间的差异有显著性意义,P＜0.05;第二组与其余各组间的差异有显著性意义,P＜0.05;第三组与第四组间的差异无显著性意义,P＞0.05.结论超声微泡造影剂在超声作用下可明显增强小鼠骨骼肌的基因转染效率;未加超声波作用时,直接肌注携基因的超声微泡造影剂亦可增加小鼠骨骼肌的基因转染效率.     

超声辐照和SonoVue微泡在介导hAng-1基因体外转染时的作用和量效关系探讨

目的 探讨超声辐照和SonoVue微泡分别使用和联用在介导hAng-1基因体外转染过程中的作用以及辐照强度和微泡浓度对转染效率和细胞活性的影响.方法 实验分四组A组:单纯超声辐照+质粒组;B组:微泡+质粒组;C组:超声辐照+微泡+质粒组和空白对照组D组. C组内转染参数分别设置为超声照射强度0.5、1.0 、1.5和2.0 W/cm~2,微泡浓度5%、10%、20%、30%和40%.将连接有eGFP-C_3-hAng-1质粒的SonoVue微泡对293T细胞进行转染,48 h后检测各组基因转染效率和细胞存活率. 结果转染48 h后C组转染效率最高,荧光阳性细胞数最多,强度最大;A组转染效率很低,见少量荧光表达;B、D组无明显基因转染发生.随着超声照射强度和微泡浓度的增加,基因转染效率会逐步升高,具有统计学意义.微泡浓度大于20%、超声照射强度超过1.5 W/cm~2后基因转染效率不再升高甚至降低,细胞死亡率显著增高(P<0.01).结论 SonoVue微泡介导外源基因转染必须联合超声辐照才能获得较好的转染效率.对于hAng-1基因和SonoVue微泡,选择声强1.5 W/cm~2,微泡浓度20%是相对最佳转染条件.     

Targeted Gene Transfection from Microbubbles into Vascular Smooth Muscle Cells Using Focused,Ultrasound-Mediated Delivery

We investigated a method for gene delivery to vascular smooth muscle cells using ultrasound triggered delivery of plasmid DNA from electrostatically coupled cationic microbubbles. Microbubbles carrying reporter plasmid DNA were acoustically ruptured in the vicinity of smooth muscle cells in vitro under a range of acoustic pressures (0 to 950 kPa) and pulse durations (0 to 100 cycles). No effect on gene transfection or viability was observed from application of microbubbles, DNA or ultrasound alone. Microbubbles in combination with ultrasound (500-kPa, 1-MHz, 50-cycle bursts at a pulse repetition frequency [PRF] of 100 Hz) significantly reduced viability both with DNA (53 ± 27%) and without (19 ± 8%). Maximal gene transfection (∼1% of cells) occurred using 50-cycle, 1-MHz pulses at 300 kPa, which resulted in 40% viability of cells. We demonstrated that we can locally deliver DNA to vascular smooth muscle cells in vitro using microbubble carriers and focused ultrasound. (E-mail: jh7fj@virginia.edu)     

不同超声强度及微泡对基因和组织作用的实验研究

目的探讨不同强度超声破坏微泡对绿色荧光蛋白质粒(green fluorcscent protein，GFP)和小鼠骨骼肌组织的作用。方法分别用0．5w／cm^2、1．0w／cm^2、2．5w／cm^2的超声作用于基因及基因和微泡的混合物2min，琼脂糖凝胶电泳观察质粒基因的电泳图谱变化。并将昆明小白鼠16只分为4组，尾静脉输入白蛋白微泡，同时分别用(0．5w／cm^2、1．0W／cm^2、2．0W／cm^2、2．5w／cm^2的超声作用于小鼠骨骼肌2min．取局部组织HE染色观察超声破坏微泡后组织显微结构的变化。结果不同能量的超声和微泡作用后GFP质粒的电泳图谱结果无变化。0．5w／cm^2超声破坏微泡后无血管充血及红细胞渗出；1．0W／cm^2超声破坏微泡后可引起约30％血管充血，极少量红细胞渗出；2．0W／cm^2超声破坏微泡后可引起约60％血管充血，红细胞渗出较明显；2．5W／cm^2的超声破坏微泡后可使部分骨骼肌变性。结论用1．0W／cm^2和2．0W／cm^2超声作用2min后引起血管充血，红细胞渗出；2．5W／cm^2超声作用2min破坏微泡后可损伤组织。1．0～2．0W／cm^2超声强度破坏微泡后对GFP质粒无明显的损害。     

Partial Restoration of Left Ventricular Systolic Function by asPLB Gene Transfer Using Ultrasound-Mediated Microbubble Destruction

In vitro and in vivo studies have demonstrated that inhibition of phospholamban (PLB) expression in myocardium can restore left ventricular systolic function in failing heart. Ultrasound mediated microbubble destruction provides a new option for noninvasive gene transfer in heart. In this study, we transfered pAAV-antisense phospholamban (pAAV-asPLB) to the hearts of myocardial infarction (MI) mice, using ultrasound mediated microbubble destruction. Then we estimated the protein levels of PLB, Ser16-PLB and cardiac sarcoplasmic reticulum Ca²⁺ ATPase (SERCA). The left ventricular ejection fraction (LVEF), fraction shortening (FS) and SERCA activity were measured as well. MI mice were generated by ligating the left anterior descending coronary artery. Microbubbles were prepared by sonicated perfluorocarbon gas with dextrose and albumin. A mixture of pAAV-asPLB plasmid and microbubble was injected via tail vein while the heart was simultaneously exposed to ultrasound via transthoracic insonation. Three weeks later, LVEF (48.2 ± 5.18% vs 39.1 ± 5.38%, p < 0.05), FS (19.6 ± 2.59% vs 16.0 ± 2.29%, p < 0.05), SERCA activity (3.00 ± 0.29 vs 2.12 ± 0.30, p < 0.05) and Ser16-PLB protein level (0.8 ± 0.25 vs 0.46 ± 0.18, p < 0.05) were increased while PLB protein level (1.45 ± 0.38 vs 2.05 ± 0.31, p < 0.05) was decreased compared with the MI mice with saline injection. The above parameters in MI mice with only pAAV-asPLB plasmid injection or pAAV-asPLB plasmid combined with ultrasound alone were not significantly improved. pAAV-LacZ was used as a reporter gene to determine the efficiency and localization of transfection. The expression of β-galactosidase was not found in liver, lung and brain, but found only in tubular epithelial cells of kidney and found in heart. These results confirm that asPLB gene transfection can be achieved by ultrasound mediated microbubble destruction with organ specificity. The effective transfection can partly restore heart function in MI mice. (E-mail: s0hu0001@hotmail.com)     

不同超声辐照时间和微泡剂量介导HepG2细胞基因转染的实验研究

目的 探讨不同超声辐照时间和微泡剂量与基因转染效率之间的关系,初步摸索适宜转染的参数条件.方法 质粒DNA用增强型绿色荧光蛋白质粒(pEGFP),以人肝癌细胞(HepG2)为研究对象.超声频率1 MHz.声强0.5 W/cm2,对加入pEGFP和不同剂量微泡的HepG2细胞辐照不同的时间,24 h后用荧光显微镜观察绿色荧光蛋白表达情况,流式细胞仪检测基因转染率,台盼蓝染色检测细胞存活率.结果 各实验组均可见不同程度荧光蛋白表达,辐照时间为20 5和微泡剂量为60μl时可达到较理想的转染效率.结论 在超声频率和声强一定的条件下,不同超声辐照时间和微泡剂量有不同的基因转染效率,20 s、60 μl是一个较理想的参数,为基因治疗进一步研究提供了参考.     

Enhancement Effect of Ultrasound-Induced Microbubble Cavitation on Branched Polyethylenimine-Mediated VEGF165 Transfection With Varied N/P Ratio

One isoform of the vascular endothelial growth factor, VEGF₁₆₅, has been reported to be a dominant mediator and regulator of angiogenic process, which plays an important role in treating cardiovascular diseases and chronically ischemic wounds. Branched polyethylenimine (bPEI) has been widely used as a non-viral delivery vector for gene therapy. Although bPEI-mediated DNA transfection efficiency can be raised by increasing the PEI nitrogen:DNA phosphate (N/P) ratio, cytotoxicity increases as well. In this study, the enhancement effect of microbubble inertial cavitation (IC) on bPEI-mediated VEGF₁₆₅ transfection was investigated, in an effort to optimize transfection efficiency using low N/P ratios. HEK 293T cells, mixed with bPEI:VEGF₁₆₅ complexes, were exposed to 1-MHz ultrasound pulses. The results show that: (1) IC activity induced by microbubble destruction can be quantified as an IC “dose” (ICD) and will increase with increasing acoustic driving pressure; (2) larger sonoporation pores can be generated by increasing ICD; (3) the transfection efficiency can be enhanced by increasing ICD until reaching a saturation level; and (4) microbubble IC activity has less cytotoxicity than bPEI, although a combinatorial effect of microbubble IC activity and bPEI could be observed on cell viability. The results suggest that, with appropriate ultrasound parameters, it is possible to optimize bPEI-mediated VEGF transfection efficiency using relatively low N/P ratios by employing ultrasound-induced microbubble inertial cavitation.     

超声造影剂Levovist介导质粒GFP转染小鼠骨骼肌细胞的实验研究

目的探讨空气型微泡造影剂Levovist介导基因转染小鼠骨骼肌细胞的作用。方法采用质粒GFP作为目的基因，超声(1MHz脉冲波，20％工作周期，空间时间峰值强度1W／cm^2)结合Levovist作用于小鼠H2K成肌细胞，照射时间分别为10s、20s、30s、40s、50S、60s，流式细胞仪测定GFP阳性细胞率，台盼蓝染色测定细胞生存率。超声结合Levovist作用于小鼠胫前肌，1周后处死小鼠，荧光显微镜测定GFP阳性肌纤维数，HE染色估计肌肉破坏面积。结果超声单独作用于H2K细胞显示出较佳的增强GFP基因表达的作用，加入微泡造影剂Levovist后，细胞死亡率显著增加，GFP基因表达水平降低；动物实验显示，Levovist结合或不结合超声均无增强GFP基因表达水平作用，但会增加肌肉损伤面积。结论本实验条件下，Levovist无增强骨骼肌细胞基因表达作用，却加重细胞损伤。     

超声介导质粒GFP转染小鼠H2K成肌细胞的实验研究

目的 探讨超声介导基因转染小鼠骨骼肌细胞的作用。方法 采用质粒GFP作为目的基因，超声(频率1MHz，脉冲波，工作周期20％)作用于H2K成肌细胞，分别使用两种空间时间峰值强度(0．5W／cm^2、1W／cm^2)，照射时间分别为10s、20s、30s、40s、50s、60s。流式细胞仪测定GFP阳性细胞率，台盼蓝染色测定细胞生存率。结果 较低能量超声(0．5W／cm^2)GFP阳性细胞率总体显著低于较高能量超声(1W／cm^2)，超声增强GFP转染H2K细胞的最佳条件为：空间时间峰值强度1W／cm^2，照射时间40～50s。超声作用并未明显增加细胞死亡率。结论 超声在增强骨骼肌细胞基因转染领域应用前景广阔。     

超声破坏微泡法介导增强型绿色荧光蛋白质粒转染兔股骨头组织的安全性及可行性(英文)

背景:超声微泡转染系统已尝试于体内多部位的基因转染,但尚未见有用于骨部位基因转染的报告。目的:观察超声破坏微泡法介导增强型绿色荧光蛋白质粒转染兔股骨头组织的效率及可行性。方法:将日本大耳白兔按随机均分为裸转染组,预照射+裸转染组,超声定位转染组,预照射+超声定位转染,重复定位转染组。其中前2组不给予超声定向转染照射,后3组利用超声微泡破裂法介导增强型绿色荧光蛋白质粒,定向基因转染兔股骨头。各组转染1周后,于荧光显微镜下观察增强型绿色荧光蛋白在股骨头组织中的表达情况。结果与结论:超声定位转染组,预照射+超声定位转染,重复定位转染组均有增强型绿色荧光蛋白表达,且复定位转染组增强型绿色荧光蛋白质粒在兔股骨头内的转染效率明显高于其他组(P<0.01)。超声定位转染组,预照射+超声定位转染,重复定位转染组兔超声照射部位软组织和骨组织切片未观察到明显损伤病灶。结果证实,超声微泡破裂法能安全、有效实现增强型绿色荧光蛋白质粒在兔股骨头组织的转染。     

Shock Waves and DNA-Cationic Lipid Assemblies: A Synergistic Approach to Express Exogenous Genes in Human Cells

Cationic lipid/DNA complexes (lipoplexes) represent a powerful tool for cell transfection; however, their use is still limited by important concerns, including toxicity and poor internalization into deep tissues. In this work, we investigated the use of shock wave-induced acoustic cavitation in vitro for the transfection of lipoplexes in human embryo kidney 293 cells. We selected shock waves with the ability to internalize 10-kDa fluorescein isothiocyanate-dextran into cells while maintaining survival rates above 50%. Cell transfection was tested using the green fluorescent protein-encoding plasmid pCX::GFPGPI2. Confocal microscopy and fluorescence-assisted cell sorting analyses revealed successful transfection after treatments ranging from 1 to 3 min using 60 to 180 shock waves at peak amplitudes of 12.3 ± 1.5 MPa. Interestingly, the combination of shock waves and lipoplexes induced a 3.1- and 3.8-fold increase in the expression of the reporter gene compared with the use of lipoplexes or shock waves alone, respectively. These results indicate that cationic DNA assembly and shock waves act in a synergistic manner to promote transfection of human cells, revealing a potential approach for non-invasive site-specific gene therapy.     

Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle

Although clinical trials of stimulation of angiogenesis by transfection of angiogenic growth factors using naked plasmid DNA or adenoviral vector have been successful, there are still unresolved problems for human gene therapy such as low transfection efficiency and safety. From this viewpoint, it is necessary to develop safe and efficient novel nonviral gene transfer methods. As therapeutic ultrasound induces cell membrane permeabilization, ultrasound irradiation might increase the transfection efficiency of naked plasmid DNA into skeletal muscle. Thus, we examined the transfection efficiency of naked plasmid DNA using ultrasound irradiation with echo contrast microbubble (Optison) in vitro and in vivo experiments. First, we examined the feasibility of ultrasound-mediated transfection of naked plasmid DNA into skeletal muscle cells. Luciferase plasmid mixed with or without Optison was transfected into cultured human skeletal muscle cells using ultrasound (1 MHz; 0.4 W(2)) for 30 s. Interestingly, luciferase activity was markedly increased in cells treated with Optison, while little luciferase activity could be detected without Optison (P < 0.01). Electron microscopy demonstrated the transient formation of holes (less than 5 microM) in the cell surface, which could possibly explain the rapid migration of the transgene into the cells. Next, we studied the in vivo transfection efficiency of naked plasmid DNA using ultrasound with Optison into skeletal muscle. Two days after transfection, luciferase activity in skeletal muscle transfected with Optison using ultrasound was significantly increased about 10-fold as compared with plasmid alone. Successful transfection was also confirmed by beta-galactosidase staining. Finally, we examined the feasibility of therapeutic angiogenesis using naked hepatocyte growth factor (HGF) plasmid in a rabbit ischemia model using the ultrasound-Optison method. Five weeks after transfection, the angiographic score and the number of capillary density in rabbits transfected with Optison using ultrasound was significantly increased as compared with HGF plasmid alone (P < 0.01), accompanied by a significant increase in blood flow and blood pressure ratio (P < 0.01). Overall, the ultrasound transfection method with Optison enhanced the transfection efficiency of naked plasmid DNA in vivo as well as in vitro. Transfection of HGF plasmid by the ultrasound-Optison method could be useful for safe clinical gene therapy to treat peripheral arterial disease without a viral vector system.     

绿色荧光蛋白标记的报告系统优化K562细胞转染条件的研究

本研究旨在提高基因转染人红白血病细胞株K562的转染效率，同时观察绿色荧光蛋白（GFP）作为报告系统用于转染条件优化的可行性。目的基因以GFP为报告系统，通过脂质体转染及电穿孔两种方式转染K562细胞，荧光显微镜下观察并计算转染效率。结果表明：电转染较脂质体转染K562细胞的效率高，电穿孔转染效率在10％左右，而脂质体转染效率小于1％。结论：电转染较脂质体转染K562细胞的效率高，GFP可作为报告系统优化K562细胞转染条件。     

不同浓度超声微泡结合共聚物P85对人肝癌细胞HepG2基因转染的影响

目的 不同浓度超声微泡造影剂(MB)结合共聚物P85后联合一定强度超声(US)辐照,初步摸索最适人肝癌细胞HepG2质粒转染及表达的MB浓度。 方法 以人肝癌细胞HepG2为研究对象,以绿色荧光蛋白(EGFP)质粒为报告基因,添加共聚物P85和不同浓度的MB后进行脉冲多普勒US辐照。24 h后台盼蓝染色评价细胞存活率,荧光显微镜和流式细胞仪评价细胞的基因转染率。 结果 MB浓度在30%时达到较理想的转染率(22.14±3.06)%,且细胞存活率无明显下降(55.73±3.32)%。添加MB后基因转染率均较pEGFP+P85+US转染率高。 结论 MB与共聚物P85结合同时给予US辐照可增强基因转染率,且在MB浓度为30%时达到较理想的转染率。     

超声辐照联合微泡转染短发卡状重组质粒诱导裸鼠宫颈癌移植瘤凋亡的研究

目的探讨超声辐照联合微泡对靶向存活素的短发卡状重组质粒（survivin—shRNA）的转染增效作用及其安全性,分析其凋亡诱导效应和增殖抑制作用。方法将荷人宫颈癌（Hela）裸鼠18只随机分为3组,每组6只。1组：质粒＋超声辐照组,注入质粒溶液后予以超声辐照;2组：质粒＋微泡＋超声辐照组,注入质粒及微泡复合物后辐照;对照组：不予任何处理。对组织样本行冰冻切片、组织学检查、转染率检测。采用免疫组化SABC法检测移植瘤增殖细胞核抗原（PCNA）及survivin蛋白在各组肿瘤标本中的表达,测定凋亡指数（AI）和增殖指数（PI）。结果2组的移植瘤内荧光表达率显著高于对照组、1组（P〈0．01）;裸鼠其他器官组织中未观察到基因表达,且未观察到明显的组织损伤;PCNA及survivin蛋白表达下降,AI明显升高,PI明显减少,与对照组及1组比较差异均有统计学意义（P〈0．05）。结论超声辐照联合微泡能显著增强基因转染效率,无明显副作用,联合Survivin—shRNA能明显诱导裸鼠体内肿瘤凋亡,抑制细胞增殖。     

Microbubble ultrasound improves the efficiency of gene transduction in skeletal muscle in vivo with reduced tissue damage

Intramuscular injection of naked plasmid DNA is a safe approach to the systemic delivery of therapeutic gene products, but with limited efficiency. We have investigated the use of microbubble ultrasound to augment naked plasmid DNA delivery by direct injection into mouse skeletal muscle in vivo, in both young (4 weeks) and older (6 months) mice. We observed that the albumin-coated microbubble, Optison (licensed for echocardiography in patients), significantly improves the transfection efficiency even in the absence of ultrasound. The increase in transgene expression is age related as Optison improves transgene expression less efficiently in older mice than in younger mice. More importantly, Optison markedly reduces muscle damage associated with naked plasmid DNA and the presence of cationic polymer PEI 25000. Ultrasound at moderate power (3 W/cm2 1 MHz, 60 s exposure, duty cycle 20%), combined with Optison, increases transfection efficiency in older, but not in young, mice. The safe clinical use of microbubbles and therapeutic ultrasound and, particularly, the protective effect of the microbubbles against tissue damage provide a highly promising approach for gene delivery in muscle in vivo.     

电穿孔法基因转染K562细胞条件的优化

目的以绿色荧光蛋白(GFP)为观察指标优化K562细胞的电穿孔基因转染条件。方法通过改变悬液体积、温度及缓冲液、质粒浓度等转染条件,采用不同条件组合后用电穿孔法将质粒DNA转入K562细胞,通过流式细胞仪和荧光显微镜观察转染效率。结果①在电压250V、电容950μF的条件下.K562细胞得到58.6%的最大转染率。②应用0.4mL细胞悬液体积,当质粒浓度≥20μg/mL时可得到较高的转染效率。③温度和缓冲液中的血清成分对电穿孔效率影响不大。结论电穿孔是一种高效的基因转染法,通过优化条件,可提高转染率。     

Ultrasound-targeted microbubble destruction enhances naked plasmid DNA transfection in rabbit Achilles tendons in vivo

The study was to investigate the probability of increasing the transfection of the gene in tendons by ultrasound-targeted microbubble destruction (UTMD), and to search for the most suitable transfection conditions. A mixture of microbubbles and enhanced green fluorescent protein (EGFP) plasmids was injected into rabbit Achilles tendons by different administration routes and the tendons were ultrasound pulse by different ultrasonic conditions in order to determine the most appropriate conditions. Then, the rabbits were divided into four groups: (1) ultrasound + microbubbles + plasmid; (2) ultrasound+ plasmid; (3) microbubble + plasmid; (4) plasmid only. EGFP expression in the tendons and other tissues, and the damage to tendon and paratenon were all observed. The results showed that EGFP expression in the tendon was higher by ultrasound pulse with 2 W cm(-2) of output intensity and a 20% duty cycle for 10 min. Local injection was determined to be the better administration route. Among the four groups, EGFP expression in Group 1 was higher than that in other groups. EGFP expression was highest on seventh day, then it gradually decrease over time, and lasted more than 56 days. EGFP expression was not found in other tissues. There was no obvious injury caused by UTMD. Under suitable conditions, it is feasible to use UTMD as a safe and effective gene transfection therapy for tendon injuries.