超声介导微泡空化增加鼠肺内皮细胞基因转染及其作用机制  

Ultrasound-mediated microbubble cavitation enhances gene transduction in rat pulmonary endothelial cells partially by affecting membrane fluidity and cytoskeleton structure

作　　者：周忠江[1] 叶海燕[2] 崔凯[1] 王玉筵[1] 

机构地区：[1]南方医科大学南方医院心内科,广东广州510515 [2]广东省人民医院妇产科,广东广州510080

出　　处：《南方医科大学学报》2010年第11期2505-2508,2515,共5页Journal of Southern Medical University

基　　金：广东省自然科学基金(05300929)

摘　　要：目的初步探讨超声介导声学微泡"空化效应"对鼠肺微血管内皮细胞基因转染的影响及其作用机制。方法 6孔板培养鼠肺微血管内皮细胞,加20μg报告基因质粒EGFP及10%白蛋白微泡(全氟显),连续波超声照射进行微泡"空化",观察不同照射时间相同机械指数(MI=1.0)(A组:30s;B组:60s;C组:90s;D组:120s;E组:180s)及不同MI相同照射时间(60s)(B1组:MI0.5;B2组:MI0.75;B3组:MI1.0;B4组:MI1.5;B5组:MI1.8)报告基因转染情况。以激光共聚焦观察细胞膜膜流动性、免疫荧光观察细胞微管蛋白及微丝蛋白。结果反应细胞膜流动性的荧光恢复强度分别为A组0.173±0.013、B组0.250±0.037、C组0.364±0.022、D组0.381±0.019、E组0.395±0.009(与A组相比,P<0.01);B1组0.171±0.017、B2组0.255±0.026、B3组0.378±0.007、B4组0.382±0.009、B5组0.397±0.008(与B1组相比,P<0.01)。反应细胞微管蛋白变化的荧光强度分别为A组159.15±4.79、B组188.23±6.20、C组205.80±4.48、D组208.99±8.34、E组213.70±5.09(与A组相比,P<0.01);B1组176.84±3.10、B2组187.57±14.52、B3组206.41±11.66、B4组220.12±13.39、B5组221.16±12.78(与B1组相比,P<0.01);各组微丝蛋白结构完整,无断裂及紊乱,荧光强度差别无统计学差异。结论超声介导微泡空化能增加基因转染率,对细胞骨架无明显损伤;当MI为1.0时,随照时延长细胞膜流动性及细胞骨架微管蛋白荧光强度增强;当照射时间为60s,随MI增高细胞膜流动性及细胞骨架微管蛋白荧光增强,但两种条件下均存在一定阈值;微丝蛋白荧光强度不随超声照射模式而改变。Objective To evaluate the effect of therapeutic ultrasound-induced microbubble＇s cavitation on plasmid gene transduction in rat pulmonary endothelial cells in relation to the changes of membrane fluidity and cytoskeleton structure.Methods Rat endothelial cells cultured in vitro were transfected with EGFP plasmid in the presence of protein microbubbles.During the transfection process,the cells were exposed to continuous 2 MHz ultrasonic irradiation for 30,60,90,120 and 180 s（groups A,B,C,D and E,respectively） with the constant mechanical index（MI） of 1.0,or for 60 s with different mechanical index（MI） of 0.5,0.75,1.0,1.5,and 1.8（groups B1,B2,B3,B4 and B5,respectively）.The changes of endothelial cytoskeletal structure and membrane fluidity were evaluated by immunofluorescence staining after the exposure.Results EGFP gene transduction increase obviously with prolonged echo irradiation and increased MI.The intensity of immunofluorescence staining,which represented endothelial membrane fluidity,was 0.173±0.013,0.250±0.037,0.364±0.022,0.381±0.019,and 0.395±0.009 in groups A-E,as compared with 0.171±0.017,0.255±0.026,0.378±0.007,0.382±0.009 and 0.397±0.008 in groups B1-B5,respectively.The recovery intensity of the immunofluorescence staining representing the changes in microtubulin of the cytoskeleton structure was 159.15±4.79,188.23±6.20,205.80±4.48,208.99±8.34,and 213.70±5.09 in groups A-E,and was 176.84±3.10,187.57±14.52,206.41±11.66,220.12±13.39 and 221.16±12.78 in groups B1-B5,respectively.The endothelial membrane fluidity and microtubule fluorescence recovery intensity increased remarkably compared with the baseline（P〈0.01） within the MI range of 0.50-1.0 and the exposure time of 30-90 s,but underwent no further changes in response to prolonged exposure time（180 s） at the MI of 1.5（P〉0.05）.No changes in microfilament fluorescence intensity were observed after exposure to different MI or irradiation time.Conclusion Therapeutic ultrasound-mediated albumin microbu

关 键 词：超声照射 微泡空化 基因转染 细胞骨架 细胞膜流动性 

分 类 号：R445.1[医药卫生—影像医学与核医学]