中国现代医学杂志2017，Vol.27Issue(6)：10-16,7.DOI:10.3969/j.issn.1005-8982.2017.06.003
人干细胞白血病基因重组慢病毒载体的构建及其在Cajal样间质细胞中的表达
Construction of recombinant human stem cell leukemia gene lentiviral vector and its expression in Cajal-like interstitial cells
摘要
Abstract
Objective To construct recombinant lentiviral vectors containing human stem cell leukemia (SCL) gene and to observe its ability in transfecting Cajal-like interstitial cells and mediating SCL gene expression. Methods The SCL gene was amplified from plasmid with SCL gene by PCR, connected to the shuttle plasmid GV287-EGFP to construct GV287-SCL, the obtained replication-defective recombinant lentiviral GV287-SCL was propagated in 293T cells according to the steps of Invitrogen Lipofectamine 2000. The recombinant lentiviral GV287-SCL was purified and titrated by dilution technique. The transfected Cajal-like interstitial cells were cultured in vitro, the distribution and efficiency of recombinant lentiviral mediated SCL were observed by the expression of green fluorescence protein (GFP) under the fluorescent microscope. RT-PCR were used to measure the expression of SCL mRNA in the transfected Cajal-like interstitial cells. Results Western blot and gene sequencing confirmed that the SCL gene was successfully inserted into the lentiviral vector, and SCL recombinant lentiviral vector was successfully transfected into Cajal-like interstitial cells. The adenovirus had a high transfection efficiency of up to 85% when MOI was 10. RT-PCR showed the expression of SCL mRNA in the Cajal interstitial cells of the experimental group was lower than that of the vector group and the blank group. Conclusions The recombinant lentiviral vector containing human SCL gene has been successfully constructed by homogenous recombination in bacteria, and it has a high transfection efficiency and can mediate expression of SCL gene in Cajal-like interstitial cells.关键词
干细胞白血病基因/慢病毒载体/转染/Cajal样间质细胞Key words
stem cell leukemia gene/lentiviral vector/transfection/Cajal-like interstitial cell分类
医药卫生引用本文复制引用
于建超,王江平,李应龙,钱彪,倪钊,王新敏,李强,王勤章..人干细胞白血病基因重组慢病毒载体的构建及其在Cajal样间质细胞中的表达[J].中国现代医学杂志,2017,27(6):10-16,7.基金项目
国家自然科学基金(No:81360120) （No:81360120）
