超声及超声微泡介导基因转染的可行性研究

目的探讨超声、超声造影剂微泡介导基因转染的可行性及所需超声照射时间。方法选择超声能量在MI=1.5、超声频率f=8MHz,向人脐静脉内皮细胞(HUVEC)悬液加入微泡后分别超声辐照20s、1、5、10、15、30min,继续培养24h。然后用台盼蓝拒染法计数活细胞,各组与对照组(无辐照)行统计学分析,选择超声辐照的最佳时长。然后,微泡与增强型绿色荧光蛋白(EGFP)质粒充分孵育后,采用该时长进行超声微泡介导基因转染试验,细胞继续培养48h后,通过荧光显微镜观察EGFP的表达。结果超声辐照10、15、30min组细胞计数比对照组降低,差异有统计学差异(P<0.05)。利用超声能量在MI=1.5、超声频率f=8MHz条件下辐照5min,继续培养48h后荧光显微镜观察,有EGFP表达,细胞生长良好。结论超声微泡能够促进质粒进入细胞并转染基因。超声能量MI=1.5、超声频率f=8MHz时,超声微泡介导基因转染的最佳辐照时长为5min。

Feasibility of gene delivery mediated by ultrasound and microbubbles

Objective To investigate the feasibility and proper exposure time of ultrasound-and microbubble-mediated gene delivery.Methods 24 h after HUVEC was exposed to ultrasound(MI=1.5,f=8 MHz) for 20 s,1 min,5 min,10 min,15 min and 30 min respectively,the number of living cells was counted by trypan blue exclusion assay to determine the best exposure time for ultrasound-and microbubble-mediated gene delivery,and then comparison was made.Then,after microbubbles and the plasmid of enhanced green fluorescent protein(EGFP) were mixed sufficiently,ultrasound-and microbubble-mediated gene delivery was carried out.After 48 h-culture,EGFP expression was detected by fluorescence microscope.Results The cells were first exposed to ultrasound with microbubbles and then after 24 h,cell number was counted.Compared with that in control group,cell count was decreased significantly in 10 min,15 min and 30 min groups(P<0.05).And 48 h after the cell was exposed to ultrasound(MI=1.5,f=8 MHz) for 5 min with microbubbles combined with EGFP plasmid,EGFP was expressed and cells grew well under the fluorescence microscope.Conclusion Ultrasound and microbubbles can promote plasmid enter the cell for gene delivery.The best exposure time for ultrasound-and microbubble-mediated gene delivery is 5 minutes when MI is 1.5 and f is 8 MHz.