pcDNA3-hNGF真核表达载体的构建及其在大鼠骨髓基质干细胞中的表达  

Construction of pcDNA3-hNGF eukaryotic expression vector and its expression in rat bone marrow stromal cells

作　　者：粟谋[1] 贝朝涌[1] 蒋林彬[2] 徐威[1] 陈宁[1] 

机构地区：[1]桂林医学院附属医院四肢创伤骨科,541001 [2]桂林医学院生物技术学院,541004

出　　处：《中国骨质疏松杂志》2016年第1期41-44,共4页Chinese Journal of Osteoporosis

基　　金：国家自然科学基金资助项目(81160223);广西自然科学基金资助项目(2015GXNSFAA139117)

摘　　要：目的构建人神经生长因子(h NGF)真核表达载体pc DNA3-h NGF,检测其在大鼠骨髓基质干细胞中的表达。方法利用PCR扩增h NGF全长c DNA,构建真核表达载体pc DNA3-h NGF,经测序证实后将其转染至大鼠骨髓基质干细胞中,利用Western blot方法检测h NGF的表达情况。结果 DNA测序结果显示构建的pc DNA3-h NGF表达载体与实验设计相符。Western blot检测显示该重组载体转染大鼠BMSCs 72h后,有h NGF的过表达。结论构建的pc DNA3-h NGF能在骨髓基质干细胞过表达h NGF蛋白,本研究为应用神经生长因子修饰的骨髓基质干细胞进行骨折治疗奠定了基础。Objective To construct an eukaryotic expression vector containing human nerve growth factor （hNGF） gene, pcDNA3-hNGF, and to examine its expression in the rat bone marrow stromal cells. Methods The full-length cDNA of hNGF was prepared using PCR, and cloned into pcDNA3 to construct the eukaryotic expression vector pcDNA3-hNGF. The vector was confirmed and then transfected into rat bone marrow stromal cells. The expression of hNGF protein was detected with Western blot. Results DNA sequencing indicated the eukaryocyte expression vector pcDNA3-hNGF was constructed as design. Western blot analysis showed that the hNGF protein was over-expressed in the rat bone marrow stromal cells after transfection with the recombinant vector for 72 hours. Conclusion The constructed eukaryocyte expression vector pcDNA3-hNGF is able to over- express hNGF protein in rat bone marrow stromal cells. This study lays the basis for the application of the bone marrow stromal cells modified with NGF in the treatment of fractures.

关 键 词：神经生长因子真核表达载体 骨髓基质干细胞 表达 

分 类 号：R68[医药卫生—骨科学]