外源性antizyme1基因转染对SH-SY5Y细胞的生物学影响

目的 研究antizyme 1(AZl)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.

Exogenous antizyme 1 gene transfection inhibits proliferation and promotes apoptosis of human neuroblastoma SH-SY5Y cells in vitro

Objective To study the effect of antizyme 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-N1,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.