摘要
基因治疗的研究已取得不少进展 ,但要成功地应用于临床仍存在许多困难 ,安全、有效的基因载体及良好的靶向性就是其中最重要的难点之一。本研究根据超声介导白蛋白微泡破裂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。实验中选择绿色荧光蛋白C3基因为标记基因 ,以白蛋白微泡为载体 ,用超声介导微泡破裂的方法在体外进行中国仓鼠卵巢细胞的基因转化 ,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。台盼蓝染色观察细胞的活性。体外试验发现 0 .8MHz、1.0W cm2 、10 %脉冲周期、60s超声介导 10 %白蛋白微泡破裂可以有效稳定转化增强的绿色荧光蛋白C3基因在中国仓鼠卵巢细胞表达 ,平均转化阳性率可达 5 6.2 % ,活性实验证明对细胞无毒副作用 。
Aim To develop a novel method to effectively deliver enhanced green fluorescence protein (EGFP)C3 into Chinese hamster orary (CHO) cells in vitro by ultrasound-mediated microbubble destruction. Methods Expression of the EGFPC3 gene was quantified by laser confocal microscopy and FACS analysis. Cell viability was assayed by Trypan Blue staining. Results Ultrasound combined with microbubbles can enhance gene transfer in cultured cells, but the effect is vary according to the utrasound condition and concetration of microbubble. Optimal gene expression occurred with microbubble at the concentration of 10% and ultrasound parameter at 0.8 MHz, 1.0 W/cm 2,10% duty cycle, 60 s. Under this condition, the transfection rate of EGFPC3 gene with ultrasound-mediated microbubble destruction method was similar to that of transfection with lipofectamine (56.2%±2.6% versus 60.8%±4.1%, P>0.05) and the relative fluorescence intensity of EGFPC3 gene was as high as that of transfection with lipofectamine (2035±32 versus 2140±28, P>0.05). Furthermore both albumin microbubble and ultrasound had no effect on cell viability. The cell viablity were 94.1%±4.6% or 93.8%±3.1% when cultured with 10% albumin microbubble or under ultrasound at 0.8 MHz, 1.0 W/cm 2, 10% duty cycle, 60 s respectively, which were no significantly difference compared with controls. Conclusions These results suggest a possible new strategy for ultrasound-mediated microbubble destruction method in gene therapy.
出处
《中国动脉硬化杂志》
CAS
CSCD
2003年第1期27-30,共4页
Chinese Journal of Arteriosclerosis
基金
江苏省自然科学基金 (BK2 0 0 2 0 2 8)资助
