中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (20): 3741-3744.doi: 10.3969/j.issn.1673-8225.2011.20.031

• 组织构建与生物活性因子 tissue construction and bioactive factors • 上一篇    下一篇

血管内皮生长因子121和骨形态蛋白2双基因共表达重组腺病毒载体的构建及鉴定

钟  声1，刘丹平1，刘素伟2，李晓禹3，李  谌1，李  媛4   

  1. 辽宁医学院附属第一医院，1骨关节外科，4神经内科三病区，辽宁省锦州市 121000；2辽宁医学院人体解剖教研室，辽宁省锦州市 121000；3鞍山市铁西医院骨二科，辽宁省鞍山市 114012
  • 收稿日期:2010-11-28 修回日期:2010-12-12 出版日期:2011-05-14 发布日期:2011-05-14
  • 通讯作者: 刘丹平，博士后，主任医师，教授，硕士生导师，辽宁医学院附属第一医院骨关节外科，辽宁省锦州市 121000 liudanping2009@sohu.com
  • 作者简介:钟声★，男，1984年生，辽宁省大连市人，汉族，辽宁医学院在读硕士，主要从事骨组织工程方面的研究。 ciguangqingran@yahoo.com.cn

Construction and identification of vascular endothelial growth factor 121 and bone morphogenetic protein-2 genes co-expressing recombinant adenovirus vector

Zhong Sheng1, Liu Dan-ping1, Liu Su-wei2, Li Xiao-yu, Li Chen1, Li Yuan4   

  1. 1Department of Bone and Joint Surgery, First Affiliated Hospital of Liaoning Medical University, Jinzhou   121000, Liaoning Province, China; 2Department of Anatomy, Liaoning Medical University, Jinzhou   121000, Liaoning Province, China; 3Second Department of Orthopaedics, West Hospital of Anshan City, Anshan   114012, Liaoning Province, China; 4Third Department of Neurology,First Affiliated Hospital of Liaoning Medical University, Jinzhou  121000, Liaoning Provine, China
  • Received:2010-11-28 Revised:2010-12-12 Online:2011-05-14 Published:2011-05-14
  • Contact: Liu Dan-ping, Doctor , Chief physician, Professor, Master’s supervisor, Department of Bone and Joint Surgery, First Affiliated Hospital of Liaoning Medical University, Jinzhou 121000, Liaoning Province, China liudanping2009@ sohu.com
  • About author:Zhong Sheng★, Studying for master’s degree, Department of Bone and Joint Surgery, First Affiliated Hospital of Liaoning Medical University, Jinzhou 121000, Liaoning Province, China ciguangqingran@yahoo.com.cn

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摘要：

背景：人血管内皮生长因子121和骨形态蛋白2在激素性股骨头坏死骨缺损中均具有成血管成骨作用，目前国内在以人血管内皮生长因子121和骨形态蛋白2基因联合治疗激素性股骨头坏死方面少有报道。
目的：构建人血管内皮生长因子121与人骨形态发生蛋白2双基因腺病毒穿梭质粒。
方法：将质粒pShuttle-CMV-VEGF121-IRES-hrGFP-1经Kpn Ⅰ/Xba Ⅰ酶切后，将BMP2片段定向连入pShuttle-CMV- VEGF121-IRES，构建可同时表达2个目的基因重组质粒pShuttle-CMV-V EGF121-IRES-BMP2，注入H5a细胞扩增，铺板，筛选阳性菌落，提取质粒，进行酶切分析及序列测定。将已构建确认正确的腺病毒质粒，经BJ5183-AD-1电转感受态细胞进行电穿孔后，铺板，筛选阳性菌落，提取质粒，进行酶切分析，PCR检测和序列分析。
结果与结论：酶切分析及核酸序列测定证实重组质粒构建正确，提示实验成功构建了血管内皮生长121及骨形态蛋白2双基因共表达重组腺病毒载体。

关键词: 人血管内皮生长121基因, 人骨形态蛋白2基因, 共表达, 腺病毒载体, 基因治疗, 基因重组

Abstract:

BACKGROUND: Vascular endothelial growth factor 121 (VEGF121) and bone morphogenetic protein-2 (BMP-2) play an important role in the development and formation of bones and vessels. At present, there are few reports about the treatment of pathogenesis of steroid-induced avascular necrosis of the femoral head (SANFH) by VEGF121 combined with BMP-2 gene in China.
OBJECTIVE: To construct VEGF121 and BMP-2 genes adenovirus shuttle plasmid pShuttle-CMV-V EGF121-IRES-BMP2.
METHODS: After Plasmid pShuttle-CMV-VEGF121-IRES-hrGFP-1 through Kpn I/Xba I, BMP-2 fragments were directionally connected to pShuttle-CMV-VEGF121-IRES. Simultaneous expression of two gene plasmid pShuttle-CMV-VEGF121-IRES was constructed, and injected with H5a cells expansion, planking, screening positive colonies, extracting plasmid, and then was undergo restriction analysis and sequencing. The correct adenovirus plasmid which has been constructed and confirmed after through BJ5183-AD-1 electroporation competent cells was undergo the electroporation, planking, screening positive colonies, extracting plasmid, and was undergo restriction analysis, PCR detection, and sequence analysis.
RESULTS AND CONCLUSION: Construction of the adenovirus shuttle plasmid was correct according to restriction analysis and nucleotide sequence detection confirmed. It is indicated that VEGF121 and BMP-2 genes co-expressing recombinant adenovirus vector can be constructed successfully in the experiment.

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