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

The experiment in the mechanisms of microbubble-mediated GFP gene enhancement in skeletal muscle in C57B10/mdx mice

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