摘要
目的探讨NEDD4-1 shRNA对U251胶质瘤细胞增殖及凋亡的影响。方法构建NEDD4-1 shRNA表达载体。以不同比例质粒和脂质体转染U251细胞,通过荧光显微镜观察细胞存活情况,优化转染效率。转染48 h后采用Western blot、RT-PCR分别检测NEDD4-1蛋白及mRNA的表达,流式细胞术检测细胞周期,MTT法检测细胞增殖,DAPI荧光染色检测细胞凋亡。结果 NEDD4-1 shRNA表达载体构建成功。脂质体与质粒的最佳转染效率比例为2.5μl∶1μg,48 h转染效率在60%～70%。与对照组比较,转染NEDD4-1 shRNA后,U251胶质瘤细胞NEDD4-1的蛋白及mRNA表达水平均下降(均P<0.05),细胞大多停滞在G0-G1期(均P<0.05),增殖率明显降低(均P<0.05),凋亡率显著增加(均P<0.05)。结论 NEDD4-1作为PTEN泛素连接酶,为胶质瘤基因治疗提供靶点。
Objective To investigate the effects of NEDD4-1 shRNA on proliferation and apoptosis of U251 glioma cells.Methods NEDD4-1 shRNA expression vector was constructed.The plasmid NEDD4-1 shRNA and liposome Lipofectamine 2000 in different proportions were transfected into U251 glioma cells,and then the cell survival was observed under fluorescence microscope.The transfection efficiency was optimized.NEDD4-1 mRNA and protein expressions in U251 glioma cells were detected by reverse transcription-polymerase chain reaction（RT-PCR） and Western blot.The cell cycle,proliferation and apoptosis were detected by flow cytometry,MTT assay and DAPI fluorescent staining respectively.Results NEDD4-1 shRNA expression vector was constructed successfully.The best proportion of NEDD4-1 shRNA and Lipofectamine 2000 for transfection efficiency was 2.5μl：1μg and the transfection efficiency was 60% to 70% 48 h after transfection.Compared with control group,the NEDD4-1 protein and mRNA expressions were decreased（all P0.05）,most of U251 glioma cells arrested in G0-G1 phase（both P0.05）,the proliferation rate of U251glioma cells was decreased（both P0.05）,and the apoptosis rate significantly increased（both P0.05） after transfection of NEDD4-1 shRNA.Conclusions NEDD4-1 as the ubiquitin ligase for PTEN is a novel target in glioma gene therapy.
出处
《中国微侵袭神经外科杂志》
CAS
北大核心
2011年第8期366-370,共5页
Chinese Journal of Minimally Invasive Neurosurgery
基金
江苏省"六大人才高峰"基金资助(编号:07-B-044)
