摘要
目的:构建人胚椎间盘髓核上皮膜蛋白-1基因低表达细胞模型,为进一步研究EMP-1在椎间盘退变中的作用机制提供理想的研究平台。方法:利用慢病毒载体介导的小发夹状RNA(small hairpin RNA,shRNA)转入人胚胎肾细胞(293FT)包装慢病毒,48h后在荧光显微镜下观察绿色荧光蛋白表达情况,72h后收集病毒上清并感染人胚椎间盘髓核细胞,Western blot、RT-PCR鉴定基因干扰效果,凝胶成像分析软件对条带显色程度进行分析,得到EMP-1/GAPDH的平均值。结果:重组慢病毒滴度测定约为1×107U/ml,感染靶细胞后得到EMP-1基因低表达细胞模型,EMP-1mRNA表达水平比正常细胞下降了50%。半定量RT-PCR结果显示EMP1/GAPDH的平均值由0.46±0.03降至0.32±0.01,Westernblot结果显示EMP1/GAPDH的平均值由0.50±0.01降至0.25±0.01。结论:通过慢病毒包装技术,成功构建人胚椎间盘髓核上皮膜蛋白-1基因低表达细胞模型。
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 lentivirus 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-1/GAPDH 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（P0.01）. Conclusion：Lentivirus packaging technology can be mastered skillfully. EMP-1 gene-deficient cell models are successfully established.
出处
《中国骨伤》
CAS
2012年第10期842-845,共4页
China Journal of Orthopaedics and Traumatology
基金
国家自然科学基金(编号:30400453)~~
关键词
椎间盘
慢病毒属
膜蛋白质类
RNA干扰
Intervertebral disk
Lentivirus
Membrane Proteins
RNA interference
