超声靶向微泡破坏技术介导报告基因对骨骼肌基因转染及不同给药途径的作用评价  

Effect of ultrasound-targeted microbubble destruction on skeletal muscle cell reporter gene transfectionby different routes of administration

作　　者：邱日想[1] 王燕莉[1] 郑婉[1] 张金山[2] 林雁[3] 陈智毅[3] 

机构地区：[1]广州医学院第三附属医院药学部,510150 [2]广州医学院第三附属医院核医学科,510150 [3]广州医学院第三附属医院超声科,510150

出　　处：《中华生物医学工程杂志》2012年第6期445-450,共6页Chinese Journal of Biomedical Engineering

基　　金：广东省自然科学基金（S2012040006593）;广东省科技厅项目（20128050300026）;广州市教育局科研项目（10A242）;广州市科信局科研项目（12C22021645）

摘　　要：目的探讨超声靶向微泡破坏（UTMD）对小鼠骨骼肌基因转染的有效性，比较不同给药途径及报告质粒转染效率的差异。方法实验随机分为单纯质粒注射组（P）、质粒＋微泡组（P＋MB）、质粒＋超声辐照组（P＋US）、质粒＋微泡＋超声辐照组（P＋UTMD）4组。将绿色荧光蛋白（GFP）质粒或红色荧光蛋白（RFP）质粒分别与微泡（或生理盐水对照）混合，将混合物分别予以局部注射（单侧鼠胫骨前肌）和尾静脉注射，进行或不进行超声辐照，辐照频率为3MHz、声强为1．0w／cm。，辐照2min，7d后处死小鼠，分别取各组小鼠的肝、心、骨骼肌组织制备冰冻切片。应用激光共聚焦显微镜检测各组小鼠骨骼肌组织荧光蛋白质粒GFP、RFP的转染效率；分析P＋UTMD组小鼠肝脏、心脏组织中的荧光素酶活性；苏木精一伊红（HE）染色观察P＋UTMD组小鼠肝、心、骨骼肌组织的病理学改变；对比P＋UTMD组小鼠局部肌肉注射和尾静脉注射时不同的给药途径下肝、心、骨骼肌组织基因转染的效果。结果单纯质粒注射组骨骼肌组织每个视野只有少数细胞表达RFP和GFP，表达率为（1．83±．1．21）％、（1．18±．0．25）％，P＋MB组和P＋US组的RFP、GFP表达率与单纯质粒注射的效果相当[（2．33±1．39）％、（3．42±1．09）％和（3．48±0．18）％、（2．52±0．33）％]；而P＋UTMD组RFP、GFP的表达率为（23．96±2．13）％、（25．69±1．98）％，显著高于其他3组（P〈0．05）。P＋UTMD组小鼠部分肝脏和心脏中均有较微弱的荧光表达，HE染色未发现UTMD对肝、心、骨骼肌组织造成明显的损伤，完整性良好，无感染、出血、水肿及细胞死亡。直接肌肉局部注射质粒联合UTMD能显著增强小鼠骨骼肌局部的基因转染，显著优于尾静脉注射（P〈0．05）。结论UTMD可有效促进基因转移，局部注射联合UTMD能显著�injection alone （group P）, injection with microbubble （group P±MB）, injection with ultrasonographic radiation （group P±US） and UTMD plus ultrasonographic radiation （group P±UTMD） respectively. Plasmids encoded with green fluorescent protein （GFP） or red fluorescent protein （RFP） were treated with microbubbles （or saline control） and injected into the tibialis anterior muscle unilaterally and caudal vein respectively. This was followed by ultrasonographic radiation （3 MHz and 1.0 W/cm2 for 2 minutes） in subgroups. The mice were sacrificed at day 7 prior to extraction of the liver, heart and skeletal muscle tissues for subsequent preparation of frozen sections. Laser confocal microscopy was employed to determine the efficiency of GFP and RFP gene transfection in skeletal muscle. Luciferase activity in the liver and heart was assayed for group P±UTMD. By using haematoxylin-eosin staining, the pathologic changes in the liver, heart and skeletal muscle were assayed for group P±UTMD. The transfection efficiency of liver, heart and skeletal muscle in group P±UTMD was compared between intramuscular and caudal vein injection respectively. Results Single plasmid injection resulted in a minority of cells showing RFP and GFP expression in skeletal muscle cells, thus yielding a rate of （ 1.83±1.21 ）% and （ 1.18± 0.25）%, respectively. Groups P＋MB and P＋US had similar expression rates of RFP [ （2.33±1.39）%, （3.48±0.18）%] and GFP [ [ （3.42±1.09）%, （2.52±0.33）%） ] when compared with group P. In contrast, a markedly higher （both P〈0.05） expression rate of RFP [ （23.96±2.13）%] and GFP [ （25.69±1.98）%] was found in group P＋UTMD, which was partly featured by slight fluorescent signals in the liver and heart tissues. Haematoxylin-eosin staining did not show evidence of significant injury of UTMD on the liver, heart and skeletal muscle, as supported by the intact tissue and absence of infection, hemorrhage, edema or cellular death. The combinati

关 键 词：超声检查 微泡 肌 骨骼 基因 报告 转染 注射 静脉内 注射 肌肉内 

分 类 号：R310[医药卫生—基础医学]