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。     

超声造影剂Optison介导质粒GFP转染小鼠骨骼肌细胞的实验研究

目的 探讨微泡造影剂Optison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(1 MHz脉冲波,20%工作周期,空间时间峰值强度1 W/cm2)结合Optison作用于小鼠体外H2K成肌细胞,照射时间分别为10、20、30、40、50及60 s,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Optison作用于小鼠胫前肌,1周后处死小鼠,荧光显微镜检测GFP阳性肌纤维数,HE染色后计算肌肉损伤面积.结果 活体外细胞实验结果显示,与阳性对照组相比,Optison结合超声作用于H2K细胞10、20及30 s时,显著增强GFP基因表达水平(P＜0.01),但于40、50及60 s时基因表达水平显著降低(P＜0.01),细胞死亡率总体显著增加(P＜0.01).动物实验结果显示,Optison单独或结合超声均显著增强GFP基因表达水平,且Optison单独作用显著减少肌肉损伤面积.结论 Optison可显著增强活体小鼠骨骼肌细胞基因表达水平,同时具有肌肉保护作用.     

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。     

纳米微泡介导GFP转染小鼠骨骼肌细胞的实验研究

目的 探讨两种纳米微泡(白蛋白外膜和磷脂外膜)增强基因转染小鼠骨骼肌的作用.方法 以正常C57B10小鼠胫前肌为研究对象,目的基因GFP与微泡混合注入小鼠胫前肌,一侧胫前肌经超声(1MHz脉冲波,脉冲重复频率为100 Hz,20％工作周期,空间峰值时间峰值声强为2 W/cm2,辐照作用时间30 s),另一侧胫前肌不经超声辐照.观察白蛋白纳米微泡及磷脂纳米微泡增强骨骼肌细胞GFP转染水平的作用.1周后处死小鼠,荧光显微镜观察发出绿色荧光者为GFP阳性肌纤维细胞,计数最大GFP阳性肌纤维细胞数,作为GFP基因转染效率指标.结果 ①白蛋白纳米微泡组和白蛋白纳米微泡+超声组最大GFP阳性肌纤维数显著高于阴性对照组(P＜0.05),显著低于阳性对照组(P＜0.05).②磷脂纳米微泡组与阴性对照组及阳性对照组比较,最大GFP阳性肌纤维数差异均无统计学意义(P＞0.05).磷脂纳米微泡+超声组最大GFP阳性肌纤维数较阴性对照组显著增高(P＜0.05);磷脂纳米微泡+超声组与阳性对照组最大GFP阳性肌纤维数差异无统计学意义(P＞0.05).结论 纳米微泡可增强基因转染骨骼肌细胞效率,白蛋白含氟化气体纳米微泡具有发展潜力.     

声诺维联合超声辐照增强绿色荧光蛋白质粒转染血管内皮细胞的实验研究

目的探讨造影剂声诺维(SonoVue)联合超声辐照人脐血管内皮细胞(HUVEC)时增强型绿色荧光蛋白报告基因质粒(pEGFP)的瞬时转染效率。方法使用连续多普勒超声辐照方式介导pEGFP转染HUVEC,照射条件为频率1.9MHz,TIS0.8,SATA为80.0mW/cm2,时间持续5min,添加或不添加2%SonoVue,pEGFP浓度为50μg/ml。48h后用流式细胞仪检测绿色荧光蛋白(GFP)瞬时转染表达率,并用台盼蓝染色检测细胞活性(生存率)。结果单纯超声辐照组GFP转染率仅为1.5%±0.2%,SonoVue联合超声辐照组为16.1%±1.9%(P<0.001);两组细胞生存率分别为94.1%±2.3%和91.1%±4.1%(P>0.05)。结论SonoVue联合超声辐照条件可增强pEGFP转染HUVEC的效率,而对细胞活性无明显影响,此方法可用于目的基因转染。     

微泡造影剂联合超声辐照介导的绿色荧光蛋白质粒转染小鼠肝癌的实验研究

目的探讨微泡造影剂SonoVue联合超声辐照在介导体内基因转染中的作用。方法建立小鼠肝癌皮下移植瘤模型，尾静脉注入绿色荧光蛋白质粒（pEGFP），添加或不添加SonoVue，脉冲多普勒超声辐照（1MHz，2W／cm^2）瘤组织。持续时间1、5、10min，7d后流式细胞仪、荧光显微镜评价pEGFP转染率。HE染色行肿瘤病理学检查。结果SonoVue联合超声辐照组pEGFP的转染率显著高于单纯超声辐照组（P〈0．01）；仅SonoVue与单纯pEGFP2组间转染率无显著差异（P〉0．05）；辐照时间5、10min时pEGFP表达明显高于1min（P〈0．05）。5与10min组间pEGFP表达无显著差异（P〉0．05）。HE染色肿瘤组织无坏死灶出现。结论微泡造影剂联合超声辐照可明显提高基因转染率，且对组织无损害。     

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.     

超声介导SonoVue微泡破裂对hAng-1基因完整性和表达效率的影响

目的 探讨超声联合SonoVue微泡介导hAng-1基因转染293T细胞的转染效率及基因完整性和表达状况.方法 构建eGFP-C3-hAn-1质粒,根据不同实验组的设计,应用相应的微泡联合超声辐照条件进行293T细胞的eGFP-C3-hAng-1基因转染,转染后48 h,以荧光显微镜观察到绿色荧光为转染成功标志;流式细胞术检测基因转染阳性细胞率,台盼蓝染色检测细胞生存率;RT-PCR和Western blot技术检测hAng-1基因的mRNA和蛋白表达;琼脂糖凝胶电泳检测经超声辐射后质粒的完整性.结果 ①微泡浓度为20%,DNA浓度为15 mg/L时进行基因转染可获较好转染效率和细胞生存率;②转染体系中血清的存在并不影响转染效率和细胞生存率;③最适转染条件下的超声辐照剂量不会影响DNA的完整性,且转染后的基因可正常表达mRNA并翻译目的蛋白.结论 微泡联合超声辐照能够介导体外细胞治疗性基因的转染,血清的存在并不影响基因的转染,转染后的基因能顺利地表达并具有正常功能.     

超声微泡介导GFP转染C57810及mdx小鼠骨骼肌细胞的实验研究

目的 探讨细胞膜孔开放及含氟烷气体白蛋白外膜在超声微泡介导GFP转染C57810及mdx小鼠骨骼肌细胞中的机制.方法 以肌细胞膜缺损为主要病理改变的mdx小鼠与正常C57810小鼠为研究对象,目的基因GFP与Optison或SonoVue混合注入小鼠胫前肌,一侧胫前肌经超声辐照,另一侧胫前肌不经超声辐照.C57810小鼠作为正常对照,实验分组如下:①C57810小鼠生理盐水组(4条左胫前肌);②C57810小鼠生理盐水+超声组(4条右胫前肌);③C57810小鼠Optison组(4条左胫前肌);④C57810小鼠Oprison+超声组(4条右胫前肌);⑤C57810小鼠SonoVue组(4条左胫前肌);⑥C57810小鼠SonoVue+超声组(4条右胫前肌).mdx肌营养不良小鼠实验分组如下:①mdx小鼠生理盐水组(4条左胫前肌);②mdx小鼠生理盐水+超声组(4条右胫前肌);③mdx小鼠+Optison组(4条左胫前肌);④mdx小鼠Optison+超声组(4条右胫前肌);⑤mdx小鼠SonoVue组(4条左胫前肌);⑥mdx小鼠SonoVue+超声组(4条右胫前肌).1周后处死小鼠,荧光显微镜观察发出绿色荧光者为GFP阳性肌纤维细胞,计数最大GFP阳性肌纤维细胞数,作为GFP基因转染效率指标.结果 正常C57810小鼠:①无超声作用时,与阴性对照组比较,Optison微泡显著提高GFP基因转染水平(P<0.01),SonoVue微泡不提高GFP基因转染水平;②有超声作用时,与阴性对照组比较,Optison微泡显著提高GFP基因转染水平(P<0.01);③有超声作用时,与阴性对照组比较,SonoVue微泡显著提高GFP基因转染水平(P<0.01).mdx小鼠:①与正常C57810小鼠比较,GFP单独(生理盐水组)显著提高GFP基因转染水平(P<0.01),Optison微泡显著提高GFP基因转染水平(P<0.01),SonoVue微泡显著提高GFP基因转染水平(P<0.01);②与阴性对照组比较,Optison微泡显著提高GFP基因转染水平(P<0.01),SonoVue微泡显著提高GFP基因转染水平(P<0.01).结论 细胞膜孔开放是微泡提高基因转染水平的重要因素,含氟烷气体白蛋白外膜是Optison微泡提高GFP转染水平的主要成分.

Abstract：

Objective To investigate the role of sonoporation and the deblic of microbubbles with perfluoropropane gas and albumin in the mechanisms of microbubble-mediated gene enhancement by experimenting in skeletal muscle in C57B10/mdx mice. Methods Plasmid DNA (10 μg) encoding green fluorescent protein (GFP) was mixed with Optison or SonoVue dissolved in saline and injected into the tibialis anterior (TA) muscle of /C57B10/mdx mice with and without adjunct ultrasound. The efficiencies of GFP transgene expression were determined under different experimental conditions. C57B10 mice as normal control:①C57B10 mice + saline (4 left TAs);②C57B10 mice + saline + ultrasound (4 right TAs) ;③C57B10 mice + Optison(4 left TAs);④C57B10 mice+ Optison + ultrasound(4 right TAs);⑤ C57B10 mice + SonoVue(4 left TAs) ;⑥C57B10 mice + SonoVue + ultrasound(4 right TAs). Mdx mice groups:① mdx mice + saline(4 left TAs) ;② mdx mice + saline + ultrasound(4 right TAs);③ mdx mice + Optison (4 left TAs) ; ④ mdx mice + Optison + ultrasound (4 right TAs); ⑤mdx mice + SonoVue(4 left TAs) ;⑥mdx mice + SonoVue + ultrasound(4 right TAs). Mice were sacrificed 1 week after plasmid DNA injection. Fibres with fluorescence green signals were determined as GFP-positive fibres by fluorescence microscopy. Readout was performed on the section with the maximum number of transfected fibers. Results C57B10 mice: ?Optison without ultrasound had significantly increased gene expression compared with negative control ( P <0. 01). SonoVue without ultrasound did not enhance gene expression. ?Optison with ultrasound had significantly increased gene expression compared with negative control (P < 0.01). ?SonoVue with ultrasound had significantly increased gene expression compared with negative control ( P<0. 01).Mdx mice:? Compared with C57B10 mice, GFP alone demonstrated significant GFP expression in mdx mice ( P <0. 01) , Optison demonstrated significant GFP expression in mdx mice ( P <0.01), and SonoVue demonstrated significant GFP expression in mdx mice ( P <0. 01). ?Microbubble groups (Optison and SonoVue) had significantly increased gene expression compared with negative control (P <0. 01). Conclusions In the mechanisms of microbubble-mediated gene enhancement, sonoporation is the key step. The deblic of microbubbles with perfluoropropane gas and albumin is the main constituent in the mechanisms of Optison-mediated gene enhancement. fibers.Results C5781 0 mice:①Optison without ultrasound had significantly increased gene expressioncompared with negative control(P<0.01).SonoVue without ultrasound did not enhance gene expression.②Optison with ultrasound had significantly increased gene expression compared with negative control(P<0.01).③SonoVue with ultrasound had significantly increased gene expression compared with negativecontr01(P相似文献   

声诺维增强超声辐照介导pEGFP-N1转染RPE细胞的实验研究

目的探讨超声与微泡造影剂声诺维（SonoVue）介导基因转染体外培养RPE细胞的作用。 方法在24孔板中,用1MHz,2W/cm2,50Hz的脉冲波辐照RPE细胞2min,质粒用量6μg/孔,加或不加20%浓度的SonoVue,72h后流式细胞仪测瞬时转染率,台盼蓝染色检测细胞活性。 结果单纯超声辐照组转染率为（1.07±0.22）%,加SonoVue组为（12.58±1.75）%（P〈0.05）,各组细胞生存率在95%以上。 结论SonoVue能促进超声辐照基因转染RPE细胞,且对细胞基本无损伤。     

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.     

共聚物P85和微泡造影剂对小鼠骨骼肌超声介导基因转染的影响

目的 探讨共聚物P85、微泡造影剂和超声在质粒DNA对小鼠骨骼肌基因转染中的影响.方法 应用共聚物P85、微泡造影剂Optison与DNA混合后直接小鼠胫前肌(TA)注射,并辐照超声.1周后取出胫前肌并快速冰冻切片,荧光显微镜计数表达GFP转染的肌纤维数,HE染色评价肌肉损伤情况.结果 共聚物P85和微泡造影剂Optison均可促进质粒DNA的基因转染(P<0.01,P<0.05).辐照超声可使P85介导的基因转染效率显著提高(P<0.01),但对微泡造影剂介导的基因转染却无显著提高(P>0.05),并且P85所介导的基因转染效率高于微泡造影剂介导的基因转染效率(P<0.01).微泡造影剂和P85耦合并辐照超声可使质粒的基因转染效率显著提高,与所有各组的差异有统计学意义(P<0.01).同时辐照超声显著增加含微泡造影剂组骨骼肌的损伤面积(P<0.01).结论 共聚物P85和微泡造影剂可介导质粒DNA的基因转染,辐照超声对其有促进作用,三者联合应用具有协同作用.     

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.     

脂质体微泡对超声介导基因转染的增效作用研究

目的 探讨超声介导基因转染时,脂质体微泡(LM)对体内、外红色荧光蛋白基因(RFP)转染的增效作用及其安全性.方法将RFP和LM加入培养的Hela细胞后行超声辐照(US),对微泡浓度、超声强度、辐照时间进行优化研究,运用荧光显微镜、流式细胞术评估基因转染率,并对细胞损伤进行分析.在裸鼠移植瘤的体内实验中,将LM和RFP质粒(P)经尾静脉注入后予以超声辐照(P+LM+US),以单纯质粒注射(P)、P+US、P+LM作为对照,行冰冻切片,组织学检查,RFP表达检测.结果培养的Hela细胞经LM和超声辐照联合处理后,RFP基因转染率显著增加,差异有统计学意义(P＜0.01),在超声强度为1.0 W/cm2、微泡浓度为6%、辐照3 min的条件下最显著,且未发现显著的细胞损伤.P+LM+US组的裸鼠移植瘤内RFP表达显著高于P组、P+US组或P+LM组,差异均有统计学意义(P＜0.01),且未观察到明显的组织损伤.结论 LM对超声介导基因转染的体内、外转染效率有显著的增效作用,而无明显的细胞或组织损伤,为临床基因治疗提供一种新颖、高效、安全的非病毒基因转染方法.