超声激励bFGF微泡促进组织细胞转染的体外实验研究  

In vitro experimental study of ultrasound-induced bFGF nano-microbubbles promoting the repair of tissue at cellular level

作　　者：陈翠兰 陈珂 黄鹂 雷芳 谢卫国 CHEN Cui-lan;CHEN Ke;HUANG Li;LEI Fang;XIE Wei-guo(Department of Ultrasound,Wuhan Third Hospital,Wuhan 430060,Hubei,China;Department of Burn,Wuhan Third Hospital,Wuhan 430060,Hubei,China)

机构地区：[1]武汉市第三医院超声科,湖北武汉430060 [2]武汉市第三医院烧伤科,湖北武汉430060

出　　处：《生物医学工程与临床》2021年第3期264-270,共7页Biomedical Engineering and Clinical Medicine

基　　金：武汉市卫生健康科研基金资助项目(WX19C24)。

摘　　要：目的利用超声激励微泡促进碱性成纤维细胞生长因子(bFGF)目的基因表达,为组织修复提供细胞水平基础。方法构建并提取b FGF目的基因质粒,其含有增强型绿色荧光蛋白(EGFP)作为标记基因,制备并鉴定含bFGF-EGFP基因微泡。将细胞成功分离、培养后,在不同实验条件下(质粒质量浓度、声强、辐照时间和占空比)进行超声激励微泡参数优化,根据每组条件下最优的细胞存活率和标记基因转染效率所对应的参数作为后续实验超声辐照条件。实验分为4组:A组为对照组,B组为单纯bFGF-EGFP质粒组,C组为超声辐照+普通脂质微泡组,D组为超声辐照+bFGF-EGFP微泡组。细胞转染24 h后,在倒置荧光显微镜下观察EGFP表达情况;转染48 h后,用流式细胞仪检测转染效率,CCK8法检测细胞存活率,逆转录聚合酶链式反应(RT-PCR)和Western blot分别检测mRNA和蛋白的相对表达量,并比较4组间的差异来评价超声激励bFGF微泡在细胞水平的生物学效应。结果携带bFGF-EGFP质粒微泡粒径大小约为(941.7±101.2) nm,Zeta电位大小约为(20.8±4.5) mV。实验研究中最优超声参数为质粒质量浓度20μg/mL,声强1.50 W/cm^(2),辐照时间60 s,占空比40%。细胞转染24 h后,A组和C组未见明显EGFP表达,B组有少量EGFP表达,而D组EGFP表达量明显增多。转染48 h后,4组细胞存活率均> 80%,各组间差异无统计学意义(P> 0.05)。流式细胞仪测量转染效率在D组[(60.0±7.9)%]较其他各组明显增加,差异有统计学意义(P <0.05)。转染48 h后,超声激励目的基因bFGF转染后D组mRNA[(45.0±5.1)%]和蛋白相对表达量[(35.0±5.0)%]均明显增高,而D组Ⅰ型胶原蛋白[(9.1±3.0)%]和Ⅲ型胶原蛋白[(11.2±3.1)%]较其他组均明显降低,差异均有统计学意义(P <0.05)。结论超声优化参数激励bFGF微泡可提高细胞转染效率并保持较高的细胞活性,促进bFGF表达效率并抑制Ⅰ型和Ⅲ型胶原蛋白;这种超声物理效应和bFGFObjective To provide a cell level basis for tissue repair, by using ultrasound stimulated microbubbles to promote targeted gene expression of basic fibroblast growth factor(bFGF). Methods The targeted gene plasmids containing enhanced green fluorescent protein(EGFP) were constructed and extracted. After the cells were successfully isolated and cultured, the parameters of the ultrasonic stimulated microbubbles were optimized under different experimental conditions(plasmid concentration, intensity of the ultrasound, irradiation time and duty cycle). The parameters corresponding to the optimal cell survival rate and transfection efficiency of the labeled genes in each group were used as the optimal conditions for the ultrasonic cavitation effect in the subsequent experiments. Four groups were set up for the study: group A as control group, group B as single bFGFEGFP plasmid group, group C as ultrasound + lipid microbubble group, group D as ultrasound + bFGF-EGFP nanobubbles group. Twenty-four hours after transfection, inverted fluorescence microscope was used to observe EGFP expression. Forty-eight hours after transfection, the cells were detected for the transfection efficiency by flow cytometry, survival rate by CCK8,m RNA and protein expression by reverse transcription polymerase chain reaction(RT-PCR) and Western blot testing, respectively, and the differences were compared within 4 groups to evaluate the biological effects of ultrasonic irradiated bFGF nanomicrobubbles at cellular level. Results The particle size of the bFGF-EGFP plasmid was(941.7 ± 101.2) nm, and the Zeta potential was(20.8 ± 4.5) mV. The optimal ultrasonic parameters in this study were: plasmid concentration 20 μg/mL, the ultrasound intensity 1.50 W/cm^(2), irradiation time 60 s, duty cycle 40 %. Twenty-four hours after transfection, no significant EGFP expression was observed in group A and group C, little was in group B, while the expression of EGFP was significantly increased in group D. Forty-eight hours after transfection, the cell su

关 键 词：超声波 碱性成纤维细胞生长因子(bFGF) 微泡 BFGF基因 物理效应 生物效应 

分 类 号：R318[医药卫生—生物医学工程] Q785[医药卫生—基础医学]