中国骨伤2012，Vol.25Issue(10)：842-845,4.DOI:10.3969/j.issn.1003-0034.2012.10.014
慢病毒介导的RNA干扰技术构建人胚椎间盘髓核上皮膜蛋白-1基因低表达细胞模型
Construction of Epithelia Membrane Protein 1 gene-deficient in human fetal nucleus pulposus cell model by lentivirus-mediated RNA interference
摘要
Abstract
Objective:To construct Epithelia Membrane Protein 1 gene-deficient in human fetal nucleus pulposus model by lentivirus-mediated RNA interference for building a platform for illustrating the biomechanisms role of EMP-1 during human intervertebral disc degeneration. Methods:The lentivirus vector with shRNA targeting EMP-1 mRNA was transected into 293FT cells by liposome. Then the lenlivirus supernatant was obtained and used for infecting human fetal nucleus pulposus. The expression of GFP was observed under fluorescence microscope after 48 h. The viral particles were collected at 72 h after transfection. The efficacy of gene interference was tested by Western blot and Real-time RT-PCR. Analysis the results of the fluorescent microscope scenes and get the average values of EMP-S/G-APDH by detected the interference efficiency of various interference DNA sequences with western blot and semi quantitative RT-PCR methods. Results: The lentivirns with high titer were obtained and the EMP-1 gene deficient cell strains were obtained. Semi quantitative RT-PCR and Western blot proved the average values of EMP-1 /GAPDH decreased from 0.46 to 0.32 and 0.5 to 0.25 (P<0.01). Conclusion: Lentivirus packaging technology can be mastered skillfully. EMP-1 gene-deficient cell models are successfully established.关键词
椎间盘/慢病毒属/膜蛋白质类/RNA干扰Key words
Intervertebral disk/ Lentivirus/ Membrane Proteins/ RNA interference引用本文复制引用
胡明,马远征,李大伟,黄凤山,杨达宇,杨同磊,刘玉川..慢病毒介导的RNA干扰技术构建人胚椎间盘髓核上皮膜蛋白-1基因低表达细胞模型[J].中国骨伤,2012,25(10):842-845,4.基金项目
国家自然科学基金(编号:30400453) （编号:30400453）
