超声介导微泡破裂法对VEGF体外转染成纤维细胞蛋白表达影响的实验研究

Experimental study on protein expression when transferring VEGF gene into the fibroblast by ultrasound-mediated microbubbles destruction in vitro

目的利用ELISA检测法观察诊断剂量的超声介导微泡破裂法对VEGF体外转染成纤维细胞蛋白表达的影响并探讨该种转染方法的最佳超声条件。方法实验分组:单纯VEGF质粒转染组;超声+造影剂微泡+VEGF质粒转染组,该组并根据MI(机械指数)分为三组1.2;1.4;1.6。转染后利用ELISA法对各组成纤维细胞的VEGF蛋白表达进行定量检测;利用锥虫蓝染色法比较不同超声强度对细胞活性的影响。结果VEGF质粒转染成纤维细胞后,24h开始即有较高水平的VEGF蛋白表达,表达量于48 h7、2 h呈下降趋势;超声+造影剂微泡+VEGF质粒组(MI1.4、1.6)细胞VEGF蛋白表达量最高,但2组间差异无显著性(P>0.05);以上2组与单纯质粒转染组及MI为1.2组比较有显著性差异(P<0.01);超声+造影剂(MI1.2)组与单纯质粒转染组亦有显著性差异(P<0.05)。锥虫蓝染色法显示:随着超声强度的增加,细胞死亡率呈上升趋势。结论诊断剂量的超声介导微泡破裂转染法能够提高VEGF体外转染成纤维细胞蛋白的表达;使用的超声剂量大,转染率高,但细胞损害大;反之,细胞损伤小,转染率低。

Objective To observe the protein expression of VEGF gene in flbroblast by diagnostic ultrasound-mediated mierobubbles destruction in vitro through enzyme-linked immunosorbent assay （ELISA） and to explore the best ultrasound conditions. Methods The cells were divided into four groups： pure VEGF plasmid; ultrasound plus contrast mierobubbles and VEGF plasmid group. The latter group is divided into three subgroups of 1.2;1.4,1.6 according to MI （mechanical index）. Protein expression and cell activity were measured quantitatively using ELISA and Trypan Blue staining after transfecting respectively. Results a level of VEGF protein showed the highest expression after 24 hours and a declining trend of the 48 h, 72 hours. VEGF protein expression was highest of the ultrasound contrast mierobubbles plus VEGF plasmid group （MI1. 4,1.6）. Compare to pure plasmid the difference was significant, but no significant difference between the two groups （ P 〉 0.05） ; there was a significant difference between above two groups and goup of ultrasound contrast mierobubbles plus VEGF plasmid group （MI1.2） （ P 〈 0.01 ） ; there was also significant difference between group （MI1.2） and pure plasmid group （ P 〈0.05）.Trypan Blue staining showed that the cell mortality take a rising trend with increasing the intensity of ultrasound. Conclusion The VEGF protein expression in fibroblast cell could be increased with the administration of diagnostic ultrasound-mediated microbubbles. The more increasing intensity of ultrasound, the higher transfecfion rate, but the more cells injury and vice versa.