超声击破微泡对重组腺病毒Ad-EGFP／HIF-1α活性的影响

Biological activity of recombinant adenovirus Ad-EGFP/HIF-1α by microbubble destruction

目的探讨超声破坏载重组腺病毒Ad—EGFP／HIF-1α的微泡对AdEGFP／HIF-1α生物学活性的影响。方法采用机械振荡法及分层吸附法制备携带重组腺病毒的脂质超声微泡造影剂，用DFY-Ⅱ型超声图像定量分析诊断仪检测微泡粒径，计算其浓度。将脐静脉内皮细胞（HUVEC）接种在24孔板中，随机分为以下5组：①空白对照组；②重组腺病毒Ad—EGFP／HIF-1α组；③载重组腺病毒Ad—EGFP／HIF-1α微泡组；④超声＋重组腺病毒AdEGFP／HIF-1α组；⑤超声＋载重组腺病毒AdEGFP／HIF-1α微泡组。荧光显微镜及流式细胞仪检测各组在细胞内的表达及转染效率，RTPCR法检测HIF-1α的mRNA的表达。结果载重组腺病毒的超声微泡的大小、粒径及分布与普通超声微泡相似。与对照组相比，各组间绿色荧光强度及转染率无明显差别；各组间HIF-1αmRNA的表达也无明显差别。结论利用超声破坏载重组腺病毒AdEGFP／HIF-1α的微泡造影剂对腺病毒活性无明显影响，为拓展超声微泡介导的基因治疗领域提供了新的思路和方法。

Objective To investigate the biological activity of recombinant adenovirus A＆EGFP/ HIF-1α by microbubble destruction in vitro. Methods The lipid ultrasound microbubbles carried with recombinant adenovirus were prepared using mechanical vibration method and stratified absorption method. The diameter and concentration of mierobubbles were determined by DFY-Ⅱ software. Human umbilical vein endothelial celI（HUVEC） was seeded in the 24-well plates, recombinant adenovirus or microbubbles carried with recombinant adenovirus was added in every well, with or without ultrasound exposure. Expression of the report gene EGFP was observed with fluorescent microscope and quantified by flow cytometry analysis. The expression of HIF-1α mRNA was detected by RT PCR. Results The average diameter and distribution of microbubbles carried with recombinant adenovirus were similar to that of common microbubbles. There was no significant difference of green fluorescence intensity, transfection efficiency and expression of HIF-1α mRNA in each group compared with the control group. Conclusions It has no significant effective on the biological activity of recombinant adenovirus after microbubble destruction, which may be taken as a potenttial way of gene therapy in future.