超声微泡转基因技术促增强型绿色荧光蛋白基因在骨缺损处转染的实验研究  

Research of enhanced green fluorescent protein gene transfer with ultrasound-mediated microbubble destruction in bone defects

作　　者：李世伟[1] 谢晓丽 杨晓东[1] 刘利君[1] 唐学阳[1] 

机构地区：[1]四川大学华西医院小儿外科,成都610041 [2]广州市妇女儿童医疗中心小儿外科,广州510623

出　　处：《中国修复重建外科杂志》2017年第4期437-442,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(81071475)~~

摘　　要：目的探讨超声微泡转基因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,不同超声辐照时间对转染效率及局部组织的影响。方法 3月龄雄性新西兰大白兔30只,体质量2.5~3.0 kg,制备右尺骨骨缺损模型,并随机分为5组(n=6)。造模后第10天于骨缺损断端间注射EGFP质粒微泡混悬液(0.3 m L/kg)后,在超声频率1 MHz、超声强度0.5 W/cm^2、占空比20%条件下,对骨缺损部位分别进行1、2、3、4、5 min超声辐照(分别为1、2、3、4、5 min组)。观察动物存活情况;转染后1周取材,大体观察骨缺损处软组织形态;荧光染色观察基因表达情况;HE染色及透射电镜观察局部组织损伤情况。结果各组动物均存活至实验完成。转染后1周各组骨缺损处有软组织生长,周围肌肉组织部分内陷填充于其间。荧光显微镜下观察,各组兔骨缺损处均有绿色荧光表达,其中2 min组表达最强,1 min组表达最弱,其吸光度(A)值与其他各组比较差异有统计学意义(P<0.05);3、4、5 min组间差异无统计学意义(P>0.05)。HE染色及透射电镜观察示,各组骨缺损处局部均有不同程度组织损伤,损伤程度随辐照时间的延长而加重。结论超声微泡转基因技术介导EGFP质粒在兔骨缺损部位转染时,其转染效率和超声辐照时间相关。当超声参数为1 MHz、0.5 W/cm^2、20%占空比时,超声辐照2 min可获得最佳转染效率及相对较轻的组织损伤。Objective To investigate the effect of ultrasonic irradiation time on enhanced green fluorescent protein （EGFP） gene transfection efficiency and local tissue in bone defects using ultrasound-mediated microbubble destruction. Methods Thirty 3-month-old New Zealand rabbits （2.5-3.0 kg in weight） were randomly divided into 5 groups （n=6） and bone defect models were made on the right ulna. At 10 days after modeling, suspension of microbubbles and EGFP plasmids were locally injected （0.3 mL/kg） and then ultrasound was performed on defect at a frequency of 1 MHz, a intensity of 0.5 W/cm2, and a duty ratio of 20% for 1, 2, 3, 4, and 5 minutes respectively （in 1, 2, 3, 4, and 5 minutes groups respectively）. The survival condition was observed. Rabbits were sacrificed for gross observation at 7 days after transfer. The gene expression was observed by fluorescence staining. HE staining and transmission electron microscopy were used to observe the local tissue damage. Results The animals all survived. New soft tissue formed in bone defects area at 1 week after transfer, the surrounding muscle tissue was partly filled in it. Green fluorescence expression was observed in all rabbits. The expression was the strongest in 2 minutes group, and was the weakest in 1 minute group. The absorbance （A） value showed significant differences when compared 1 minute and 2 minutes groups with other groups （P〈0.05）, but no significant difference was found between 3, 4, and 5 minutes groups （P〉0.05）. Tissue damage was observed in all groups and it was aggravated with the increase of irradiation time. Conclusion EGFP transfection efficiency in bone defect by ultrasound-mediated microbubble destruction is related to irradiation time. EGFP gene can be efficiently transfected without obvious toxicity at 1 MHz, 0.5W/cm2, and duty ratio of 20% for 2 minutes in bone defects of rabbits.

关 键 词：骨缺损 超声微泡转基因技术 基因转染 增强型绿色荧光蛋白 兔 

分 类 号：R683[医药卫生—骨科学]