pcDNA3．1-血管内皮生长因子165质粒局部注射对大鼠软组织缺损修复的影响  

Local injection of pcDNA3.1-vascular endothelial growth factor 165 plasmid affects the repair of rat soft tissue defects

作　　者：杨加峰[1] 武士清[2] 赵冬梅[2] 刘军莉[3] 邱丽萍[1] 王海斌[2] 许复郁[4] 

机构地区：[1]山东大学第二医院整形科,山东大学组织工程研究所,山东大学材料液态结构及其遗传性教育部重点实验室,山东省济南市250033 [2]山东大学第二医院骨外科,山东大学组织工程研究所,山东省济南市250033 [3]山东大学第二医院分子生物学实验室,山东省济南市250033 [4]山东大学第二医院病理科,山东省济南市250033

出　　处：《中国组织工程研究与临床康复》2008年第20期3901-3904,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：山东省科技攻关计划(032050112);血管内皮细胞生长因子基因修复骨缺损的实验研究山东省自然科学基金(Q2006C08);兔耳CTGF基因测序及RNAi阻断其表达治疗兔耳增生性瘢痕的研究山东省卫生厅项目(2003);血管内皮细胞生长因子基因修复软组织损伤的实验研究~~

摘　　要：背景:目前关于血管内皮生长因子(vascular endothelial growth factor,VEGF)在创伤及整形外科领域主要集中在骨折及组织工程化材料等方面,在软组织损伤方面报道不多。目的:构建的pcDNA3.1-VEGF165质粒,直接注射于大鼠软组织缺损局部,探讨其对软组织缺损修复的影响。设计、时间及地点:于2004-09/2006-01在山东大学动物实验中心和分子生物学实验室完成自身对照观察实验。材料:健康SD大鼠30只。方法:Trizol法提取兔组织中总RNA,反转录合成VEGF165cDNA序列,将此基因片段与pMD18-T构建真核表达载体,双酶切,在T4DNA连接酶作用下,再与pcDNA3.1载体构建成重组真核表达质粒pcDNA3.1-VEGF165;采用SD大鼠30只,于脊背后部两侧对称部位制备圆形全层皮肤(直径15mm)、肌肉(切除2mm厚肌肉)软组织缺损,两创缘相距4cm;选择一侧为实验组,直接注射0.2mL(含200ng)质粒液,对侧为对照组,以等量生理盐水同法处理。主要观察指标:创面愈合、血管生成及局部胶原表达。结果:30只均进入结果分析。①经赵冬梅等的方法鉴定,pcDNA3.1-VEGF165质粒构建成功。②质粒液局部注射后实验组创面愈合时间早于对照组,差异有显著性意义(P<0.05);实验组术后7,14d微血管密度高于对照组,差异有显著性意义(P<0.05)。③实验组3d即有Ⅲ型胶原表达,14d达高峰,30d时略有降低。对照组Ⅲ型胶原表达部位和趋势与实验组一致,只是时间延迟。结论:pcDNA3.1-VEGF165质粒局部注射可促进软组织缺损区血管生成和细胞外基质合成,加速创面愈合。BACKGROUND： Present studies on vascular endothelial growth factor （VEGF） are mainly on fracture and tissue-engineered materials in trauma and plastic surgery field, but few on soft tissue injury. OBJECTIVE： Constructed pcDNA3.1-VEGF165 plasmid was directly injected into local defected rat soft tissues. This study aimed to investigate its effect on defected soft tissues. DESIGN, TIME AND SETTING： A own control observational study was performed at the Animal Experimental Center and Laboratory of Molecular biology of Shandong University from September 20004 to January 2006. MATERIALS： A total of 30 healthy SD rats were selected in the experiment. METHODS： Total RNA was extracted from rabbits by Trizol method. VEGF165 cDNA sequence was synthesized by reverse transcription, which was connected with pMD18-T to construct eukaryotic expression vector, subsequently, with pcDNA3.1 to construct recombinant eukaryotic expression plasmid pcDNA3.1-VEGF165 after treated with T4 DNA ligase. Round wound （diameter 15 mm） was made on the back of 30 SD rats. The skin and 2 mm muscle were removed. There was a 4 cm distance between two wounds. One side served as an experimental group was treated with 0,2 mL plasmid liquid （containing 200 ng）, Another side served as control received saline of the same volume. MAIN OUTCOME MEASURES： Wound healing, vascularization and local collagen expression, RESULTS： A total of 30 rats were included in the final analysis, The pcDNA3,1/VEGF165 vector was constructed successfully according to the methods made by Zhao, Wound healing time was shorter in the experimental group than in the control group after local injection （P 〈 0.05）, Microvessel density （MVD） was higher in the experimental group than in the control group 7 and 14 days after surgery （P 〈 0.05）. Type I collagen could be detected at day 3, and reached a peak at day 14, and then decreased at day 30 in the experimental group. It was the same as the control group, but time delay. CONCLUSION

关 键 词：基因转染 血管内皮细胞生长因子 胶原 免疫组织化学 

分 类 号：R392.114[医药卫生—免疫学]