人血管生成素1真核表达载体的构建及在兔骨髓间充质干细胞的表达  

CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR FOR HUMAN ANGIOPOIETIN 1 AND ITS EXPRESSION IN THE MARROW MESENCHYMAL STEM CELLS OF RABBIT

作　　者：张晔[1] 曾炳芳[1] 张长青[1] 王建华[1] 

机构地区：[1]上海交通大学附属第六人民医院骨科,上海200233

出　　处：《中国修复重建外科杂志》2005年第5期377-380,共4页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：上海市科委重点基金资助项目 (0 1 JC1 4 0 0 5)~~

摘　　要：目的　探索构建人血管生成素1( human angiopoietin 1,h Ang- 1)真核表达载体,转染体外培养兔骨髓间充质干细胞( marrow mesenchymal stem cells,MSCs)修复骨缺损的可能性。　方法　基因重组和限制性内切酶酶切构建真核表达载体pc DNA3- h Ang- 1,经脂质体DOTAP介导质粒转染兔MSCs,G4 18筛选阳性克隆,RT- PCR及免疫印迹杂交检测h Ang- 1m RNA及蛋白表达。　结果　重组真核表达载体pc DNA3- h Ang- 1经限制性内切酶Xho- I和Bam H- I酶切后,电泳显示1.4 kb h Ang- 1目的片段和5 .4 kb pc DNA3载体片段,转染兔MSCs后,RT- PCR检测出目的基因m RNA,免疫印迹杂交检出h Ang- 1的蛋白表达。　结论　构建的真核表达载体pc DNA3- h Ang- 1能在转染的兔MSCs中表达。Objective To investigate the possibility of constructing eukaryotic expression vector for human angiopoietin 1(hAng 1),transfecting it to bone marrow mesenchymal stem cells (MSCs) so as to repair bone defect. Methods The eukaryotic expression vector pcDNA3 hAng 1 was constructed by recombinant DNA technique, transfected into MSCs by liposome DOTAP, and selected with G418. The hAng 1 expression of mRNA and protein was detected by reverse transcript PCR and Western Blot. Results After the recombinant eukaryotic expression vector for hAng 1 was digested with Xho I and BamH I, electrophoresis revealed 1 4 kb fragment for hAng 1 gene and 5.4 kb fragment for pcDNA3 vector. In the transfected MSCs, the mRNA and protein expression of hAng 1 gene were detected with reverse transcript PCR and Western Blot. Conclusion The constructed eukaryotic expression vector hAng 1 could be expressed in the transfected MSCs, thus to provide the basis for bone repair with tissue engineering.

关 键 词：骨髓间充质干细胞 人血管生成素 ANGIOPOIETIN 重组真核表达载体 RT-PCR检测 限制性内切酶 兔 MSCs pcDNA3 基因mRNA 免疫印迹杂交 蛋白表达 DOTAP RTPCR 体外培养 基因重组 阳性克隆 杂交检测 组织工程 转染 可能性 骨缺损 

分 类 号：R318.0[医药卫生—生物医学工程]