腺病毒介导人TGF-β_1基因转染自体腘绳肌腱重建兔前交叉韧带术后的表达  

EXPRESSION OF HUMAN TR ANSFORMING GROW TH FACTOR β_1 GENE MEDIATED BY ADENOVIRUS IN HAMSTRING TENDON AFTER ANTERIOR CRUCIATE LIGAMENT RECONSTRUCTION IN RABBITS

作　　者：王晓旭[1] 何敏[1] 谭文甫[1] 谭光华[1] 杨俊涛[1] 洪亮[1] 

机构地区：[1]南华大学附属第二医院关节外科,湖南衡阳421001

出　　处：《中国修复重建外科杂志》2016年第10期1233-1237,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：2013年湖南省教育厅资助项目(13C836)~~

摘　　要：目的 检测腺病毒介导人TGF-β_1（human TGF-β_1,h TGF-β_1）基因转染至腘绳肌腱重建兔前交叉韧带（anterior cruciate ligament,ACL）术后不同时间点的表达。方法 以DMEM培养基稀释重组腺病毒载体Adh TGF-β_1与腺病毒空载体Ad-绿色荧光蛋白（green l uorescent protein,GFP）,使病毒滴度为5×10~8 PFU/m L。取15~20月龄雄性新西兰大白兔48只,随机分为A、B、C 3组（n=16）,以兔双侧后肢膝关节为研究对象,取自体腘绳肌腱重建同侧ACL,重建前A、B组腘绳肌腱分别以Ad-h TGF-β_1及Ad-GFP转染12 h,C组以DMEM培养作为对照组。转染后12 h应用荧光显微镜观察A、B组绿色荧光表达,ELISA法检测A组腘绳肌腱中TGF-β_1蛋白表达量。术后2、4、6、8周取材,应用实时荧光定量PCR及Western blot法检测腘绳肌腱中h TGF-β_1基因及蛋白表达。结果 A、B组腘绳肌腱经转染后均可见绿色荧光表达;ELISA法检测示,A组腘绳肌腱中TGF-β_1蛋白表达量为（221.0±12.2）ng/m L。实时荧光定量PCR检测示,术后各时间点B、C组均未能检测到h TGF-β_1 m RNA表达;术后2、4、6、8周,A组h TGF-β_1 m RNA相对表达量逐渐下降,分别为1.004±0.072、0.785±0.038、0.469±0.053、0.172±0.021,各时间点间比较差异均有统计学意义（P〈0.05）。Western blot检测示,术后各时间点B、C组TGF-β_1蛋白条带均为弱阳性,A组TGF-β_1蛋白表达量均显著高于B、C组（P〈0.05）,B、C组间比较差异无统计学意义（P〉0.05）。A组TGF-β_1蛋白表达量随时间延长逐渐降低,各时间点间比较差异均有统计学意义（P〈0.05）。结论 在腺病毒载体介导下,h TGF-β1基因能成功转染至腘绳肌腱并能在ACL重建术后有效表达。Objective To detect the expression of human transforming growth factor β1 （hTGF-β1） gene mediated by adenovirus （Ad） in hamstring tendon after anterior cruciate ligament （ACL） reconstruction in rabbits. Methods Ad- hTGF-β1 and Ad-green fluorescent protein （GFP） were diluted to 5x108 PFU/mL with DMEM. Forty-eight New Zealand white rabbits were divided into 3 groups randomly （n=16） for ACL reconstruction with hamstring tendon autograft. Hamstring tendon was cultured and transfected with Ad-hTGF-β1 （group A） and Ad-GFP （group B） for 12 hours before ACL reconstruction, and was cultured with DMEM in group C. After 12 hours of transfection, green fluorescence was observed in groups A and B under fluorescence microscopy. At 2, 4, 6, and 8 weeks after operation, the hamstring tendon was harvested to detect the mRNA and protein expressions of hTGF-β, by real time fluorescence quantitative PCR and Western blot. Results Green fluorescence was observed after 12 hours of transfection in groups A and B. TGF-β, protein level reached （221.0±12.2） ng/mL at 12 hours in group A. The hTGF-β1 mRNA expression could be detected in group A, but it could not be detected in group B and group C. The mRNA expression levels of hTGF-[~, were 1.004±0.072 at 2 weeks, 0.785±0.038 at 4 weeks, 0.469±0.053 at 6 weeks, and 0.172±0.021 at 8 weeks in group A, showing significant difference （P〈0.05）. Western blot results showed weakly positive band in groups B and C; the protein expression of TGF-β1 in group A was significantly higher than that in groups B and C （P〈0.05）, but no significant difference was found between groups B and C （P〉0.05）. The protein expression of TGF-β1 gradually reduced with time, showing significant difference between different time points （P〈0.05）. Conclusion Ad-hTGF-β1 can transfect the hamstring tendon successfully, and it can effectively express for a long time after ACL reconstruction.

关 键 词：人TGF-β_1 腺病毒 腘绳肌腱 前交叉韧带 兔 

分 类 号：R687[医药卫生—骨科学]