新型白蛋白微泡经超声介导破裂促进EGFP基因在Cos-7细胞的表达

A kind of new albumin microbubble enhances EGFP gene expression in Cos-7 cell by ultrasound-mediated microbubble destruction

目的 根据超声介导白蛋白微泡破裂空化效应可以增加真核细胞膜对大分子 (如DNA)通透性的原理 ,探讨一种新的转基因方法 ,以便安全有效和定向地转移目的基因。方法 实验中选择绿色荧光蛋白基因EGFP为标记基因 ,以自制的白蛋白微泡为载体 ,用超声介导微泡破裂的方法在体外进行Cos 7细胞的基因转化 ,同时以脂质体为对照 ,激光共聚焦显微镜和流式细胞计数仪分别定性和定量观察细胞转化效率。锥虫蓝染色观察细胞的活性。结果 体外试验发现 0 .8MHz、1.0W /cm2 、10 %占空比 (dutycycle)、60s超声介导 10 %白蛋白微泡破裂可以有效稳定地转化EGFP基因在Cos 7细胞表达 ,且对细胞无毒副作用。结论 自制白蛋白微泡是一种安全、有效的新型基因载体 ,在一定超声条件控制下 ,能增强基因的转导与表达 ,有良好的靶向性 ,提示该技术有应用于临床基因治疗的广阔前景。

ObjectiveTo find a new approach of transfecting the objective gene safely and effectively according to the cavitation effect of ultrasound mediated microbubble destruction which could increase the permeability of macromolecule (such as DNA) across the eucell membrane.MethodsEGFP gene was transfected to Cos 7 cell as mark one in vitro by ultrasound mediated microbubble destruction and liposome as control. The transfection effect was surveyed by laser microscope and flow cytometry qualitively and quantitively. Trypan blue staining was adopted to measure the cell vitality.ResultsUltrasound mediated microbubble destruction at 0.8 MHz , 1.0 W/cm 2, 10% duty cycle, 60 s can get the most stable effective expression of EGFP gene in Cos 7 cell and no cytotoxicity. ConclusionsAlbumin microbubble made by us is a new and effective carrier of objective gene in gene therapy. At some specific ultrasound condition, microbubble destruction can enhance the objective gene transfection and expression and have a good targetivation. Ultrasound mediated destruction of albumin coated microbubble is a promising method in gene therapy.