不同途径注射质粒微泡对超声微泡破碎技术介导EGFP基因在兔骨缺损处转染的影响  

Effect of different injection routes of plasmid-microbubble suspension on EGFP gene transfection in bone defect using ultrasound-mediated microbubble destruction

作　　者：李世伟[1] 杨晓东[1] 唐学阳[1] LI Shiwei;YANG Xiaodong;TANG Xueyang(Department of Pediatric Surgery,West China Hospital,Sichuan University,Chengdu 610041,China)

机构地区：[1]四川大学华西医院小儿外科,成都610041

出　　处：《山西医科大学学报》2023年第4期493-497,共5页Journal of Shanxi Medical University

基　　金：四川省科技厅重点研发项目(2019YFS0265)。

摘　　要：目的探讨超声微泡破碎技术介导增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因在兔骨缺损处转染时,不同途径注射质粒微泡混悬液对转染效率及局部组织的影响。方法3月龄新西兰大白兔10只,制备右尺骨骨缺损模型,按照随机数字表法分为静脉组和断端间组(n=5)。静脉组和断端间组造模后第10天分别经耳缘静脉或骨缺损断端间向兔体内注射携带EGFP基因的质粒微泡混悬液(0.3 ml/kg)。在超声频率1 MHz,超声强度1.0 W/cm^(2),占空比20%条件下,对两组骨缺损部位超声辐照1 min,进行EGFP基因转染。在基因转染后1周时处死兔,于骨缺损处获取标本制作切片,荧光染色观察各组EGFP表达情况。采用病理图像分析软件分析计算平均光密度。HE染色观察断端间软组织病理特点。结果静脉组和断端间组均观察到绿色荧光蛋白表达。断端间组平均光密度高于静脉组,差异有统计学意义(0.0345±0.0028 vs 0.0004±0.0001,P<0.05)。表达绿色荧光蛋白的细胞主要为骨骼肌细胞,各组未见细胞坏死征象。结论超声微泡破碎技术介导EGFP基因在兔骨缺损处转染时,其效率受不同途径注射质粒微泡混悬液的影响,骨缺损断端间直接注射优于静脉注射。Objective To investigate the effect of different injection routes of plasmid-microbubble suspension on enhanced green fluorescence protein(EGFP)gene transfection in tissue between bone defects by ultrasound-mediated microbubble destruction.Methods Bone defect models were generated on the right ulnas of ten 3-month-old New Zealand rabbits,and then they were divided into intravenous injection group and local injection group by means of random number table,with five in each group.At day 10 after model generation,the suspension of plasmids carrying EGFP gene and microbubbles(0.3 ml/kg)was injected via ear vein in intravenous injection group and injected at bone defect sites in local injection group(0.3 ml/kg).Ultrasound was performed at bone defect sites at a frequency of 1 MHz,an intensity of 1 W/cm^(2),and a duty ratio of 20%for one min to mediate the gene transfection.All rabbits were sacrificed for sample harvesting at day 7 after gene transfection.The EGFP expression was quantified by average optical density under a fluorescence microscope.Pathological features of tissue damage were observed under an optical microscope.Results Green fluorescence protein was observed in both two groups.The average optical density was significantly higher in local injection group than that in intravenous injection group(0.0345±0.0028 vs 0.0004±0.0001,P<0.05).Green fluorescence protein was mainly expressed in skeletal muscle cells.No signs of cell necrosis were found in each group.Conclusion The transfection efficacy is better by injection of suspension of EGFP plasmids and microbubbles at bone defect site than that of intravenous injection.

关 键 词：超声微泡破碎技术 增强型绿色荧光蛋白 基因治疗 骨缺损 新西兰大白兔 

分 类 号：R683[医药卫生—骨科学]