超声微泡介导GFP转染C57810及mdx小鼠骨骼肌细胞的实验研究

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

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Optison＋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，含氟烷气体自蛋白外膜是Optison微泡提高GFP转染水平的主要成分。

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 normalcontrol：（1）C57B10 mice ＋ saline（4 left TAs）;（2）C57B10 mice ＋ saline ＋ ultrasound（4 right TAs） ;（3）C57B10 mice ＋ Optison（4 left TAs） ;（4）C57B10 mice ＋ Optison ＋ ultrasound（4 right TAs） （5） C57B10 mice ＋ SonoVue（4 left TAs） ;（6）C57B10 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）;（5）mdx mice ＋ SonoVue（4 left TAs） ;（6）mdx 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：（1）Optison without ultrasound had significantly increased gene expression compared with negative control （ P 〈0.01）. SonoVue without ultrasound did not enhance gene expression. （2） Oprison with ultrasound had significantly increased gene expression compared with negative control （P 〈 0.01）. （3）SonoVue with ultrasound had significantly increased gene expression compared with negative control （P 〈0.01）. Mdx mice： （1）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）. （2）Microbubble groups （Optison and SonoVue） had significantly increased gene expression compared with negative control （P 〈0.01）. Conclusions In the mechanisms of mierobubble-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.