Jiang Li, Ma Wen-li, Li Jin, Peng Yi-fei, Xu Bing, Zheng Wen-ling
Institute of Genetic Engineering, Southern Medical University, Guangzhou 510515, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2007 Nov;27(11):1709-13.
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.
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.
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.
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.
研究抗酶1（ZA1）基因转染对人神经母细胞瘤SH-SY5Y细胞体外增殖、细胞周期及凋亡的影响。
将突变型AZ1基因克隆至载体pEGFP-N1构建重组真核表达载体pAZ1m，随后转染至SH-SY5Y细胞。采用MTT法检测转染后细胞的增殖情况，运用流式细胞术分析细胞周期及凋亡的变化。进行RT-PCR检测细胞周期蛋白D1（cyclin D1）和半胱天冬酶-3（caspase-3）mRNA表达，采用蛋白质免疫印迹法检测cyclin D1蛋白表达，使用caspase-3检测试剂盒检测caspase-3活性变化。
AZ1基因转染显著抑制SH-SY5Y细胞的增殖，使细胞周期阻滞于G0/G1期，下调cyclin D1表达，上调caspase-3表达。转染细胞中还观察到caspase-3活性明显增加。
外源性AZ1基因转染可通过下调cyclin D1表达抑制SH-SY5Y细胞的增殖并导致细胞周期阻滞。AZ1基因转染导致的caspase-3表达上调可能诱导神经母细胞瘤细胞凋亡。
