摘要
目的研究antizyme1(AZ1)基因对成人神经廇SH-SY5Y细胞增殖、细胞周期及凋亡的影响。方法将构建好的AZ1基因重组真核表达载体pAZ1m稳定转染SH-SY5Y细胞。通过MTT法检测细胞增殖变化,流式细胞术分析AZ1转染对细胞周期及凋亡的影响。RT-PCR与Western blotting检测AZ1基因转染对cyclin D1和caspase-3表达的影响,caspase-3试剂盒检测酶活性变化。结果稳定转染SH-SY5Y细胞后,检测结果显示AZ1基因转染能够减慢SH-SY5Y细胞增殖速度,并使细胞停滞于G0/G1期。在cyclin D1基因表达抑制的同时,caspase-3基因表达上调。酶活性测定显示Caspase-3活性上升。结论AZ1基因能够抑制SH-SY5Y细胞增殖,通过降低cyclin D1的表达阻滞细胞周期于G0/G1期,并上调caspase-3表达促进SH-SY5Y细胞凋亡。
Objective To study the effect ofantizyme 1 （ZA1） gene transfection on the cell proliferation, cell cycle and apoptosis of human neuroblastoma SH-SY5Y cells in vitro, Methods The recombinant eukaryotic expression vector pAZ1m was constructed by cloning mutant AZ1 gene into the vector pEGFP-NI, and subsequently transfected in SH-SY5Y cells, The transfected cell proliferation was examined using MTT assay, and the changes in cell cycle and apoptosis were assayed using flow cytometry analysis, RT-PCR was performed to measure cyclin D1 and caspase-3 mRNA expressions, Western blotting carried out to examine cyclin D1 protein expression, and the changes in caspase-3 activity were detected using a caspase-3 detection kit, Results AZ1 gene transfection significantly inhibited the proliferation of SH-SY5Y cells, causing cell cycle arrest at G0/G1 stage and down-regulated cyclin D1 and up-regulated caspase-3 expressions. Obviously increased caspase-3 activity was also observed in the transfected cells. Conclusion Exogenous AZ1 gene transfection can inhibit the proliferation and cause cell cycle arrest of SH-SY5Y cells by down-regulating cyclin D1 expression. Up-regulated caspase-3 expression resulting from AZ1 gene transfection may induce apoptosis of the neuroblastoma cells.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2007年第11期1709-1713,共5页
Journal of Southern Medical University
基金
广东省自然科学基金(5004737)~~
关键词
抗酶
细胞增殖
细胞周期
凋亡
antizyme
cell proliferation
cell cycle
apoptosis
