摘要
目的:构建人IL-1Ra真核表达载体,体外转染大鼠骨髓间充质干细胞(BM-MSCs)。方法:从PHA活化的人外周血单个核细胞中提取总RNA,RT-PCR法获得hIL-1Ra cDNA,经HindⅢ、EcoRⅠ双酶切,定向克隆到真核表达载体pcD-NA3。经酶切分析及PCR鉴定后,脂质体介导下体外转染大鼠BM-MSCs,免疫荧光法检测hIL-1Ra在大鼠MSCs的表达。结果:获得570 bp大小的IL-1Ra cDNA片段;经酶切分析、PCR鉴定及DNA测序,证实IL-1Ra cDNA定向插入pcDNA3质粒,目的序列与GenBank源序列完全一致;脂质体介导转染48h后,免疫荧光检测到hIL-1Ra在大鼠MSCs的表达。结论:成功构建pcDNA3-hIL-1Ra真核表达载体并在大鼠BM-MSCs中表达,为进一步的hIL-1Ra基因修饰MSCs研究提供了实验基础。
Objective：To construct eukaryotic expression vector encoding human IL - 1 receptor antagonist （hIL - 1Ra） gene, and transfect it into rat bone marrow mesenchymal stem ceils （ BM - MSCs） in vitro. Methods： Total RNA was extracted from PHA -stimulated health human peripheral blood mononuclear ceils. The cDNA of human IL - 1Ra was got by RT - PCR, and was inserted into eukaryotic expression vector pcDNA3 after digested with Hind m and EcoR I ~ After confirmed by restriction analysis, PCR amplification and DNA sequencing, the recombinant plasmid pcDNA3 - hIL - 1Ra was transfected into rat BM - MSCs via Lipofectamine in vitro, and then the expression of hIL - 1Ra in the rat MSCs was detected by immunofluorescence. Results： The size of RT - PCR products was 570 bp as expected, and after restriction analysis, PCR amplification and DNA sequencing, the pcDNA3 - hIL - 1Ra was found to be well constructed. The expression of hIL - 1Ra in rat BM - MSCs was found by immunofluorescence at 48 h after transfection. Conclusion：The eukaryotic expression vector encoding human IL - 1Ra gene has been constructed and transfected into MSCs in vitro successfully, which might provide a basis for further investigation of applying hIL - 1Ra in clinics.
出处
《西北国防医学杂志》
CAS
2007年第6期431-433,共3页
Medical Journal of National Defending Forces in Northwest China
关键词
IL-1RA
基因转染
干细胞
骨髓
间充质
IL - 1 receptor antagonist
Gene transfection
Stem ceils, myeloid
Mesenchyme
