鸟氨酸脱羧酶抗酶融合蛋白高表达对小鼠黑素瘤细胞B16-F1细胞周期的影响

Over-expression of ornithine decarboxylase antizyme fusion proteins affects the cell cycle of mouse melanoma B16-F1 cells

目的研究鸟氨酸脱羧酶抗酶(OAZ)与绿色荧光蛋白融合蛋白GFP-OAZ1和GFP-OAZ2高表达对小鼠黑色素瘤B16-F1细胞周期的影响。方法构建GFP-OAZ1和GFP-OAZ2融合基因的真核表达质粒并经脂质体法瞬时转染B16-F1细胞,然后用Western blot分析法和荧光显微镜下观察融合蛋白在B16-F1细胞中的表达。流式细胞分析用于检测GFP-OAZ融合蛋白高表达对B16-F1细胞周期的影响。Western blot分析法鉴定GFP-OAZ融合蛋白高表达对B16-F1细胞中鸟氨酸脱羧酶(ODC)酶蛋白水平的影响。结果成功构建的GFP-OAZ1和GFP-OAZ2融合基因B16-F1细胞中正确高效表达。经流式细胞检测发现,OAZ1和OAZ2融合蛋白高表达导致细胞G0/G1期阻滞。当用OAZ1和OAZ2分别与ODC共转染B16-F1细胞时,OAZ1融合蛋白高表达显著性减低细胞内ODC蛋白水平,但OAZ2融合蛋白高表达无此种影响。结论成功构建GFP-OAZ1和GFP-OAZ2融合基因的真核表达载体,OAZ1和OAZ2融合蛋白高表达均能将B16-F1细胞阻滞于G0/G1期,但仅OAZ1融合蛋白能显著性促进ODC降解,OAZ2融合蛋白无此种功能。

This study aims to assess the effects of over-expressed GFP-OAZ1（green fluorescent protein-ornithine decarboxylase antizyme-1） and GFP-OAZ2（green fluorescent protein-ornithine decarboxylase antizyme-2） fusion proteins on cell cycle of mouse melanoma B16-F1 cells.GFP-OAZ1 and GFP-OAZ2 fusion genes were constructed truly,then transiently transfected into B16-F1 cells by lipofectamine reagent.The expressions of GFP-OAZ1 and GFP-OAZ2 fusion proteins were confirmed by immunocytochemistry and Western blot analysis.Flow cytometry was applied to detect the effect of the fusion proteins on the cell cycle of B16-F1 cells;Western blot analysis was also used to detect the effect of GFP-OAZ1/2 on ornithine decarboxylase（ODC） in protein level.Results showed that over-expression of GFP-OAZ1 and GFP-OAZ2 fusion proteins in B16-F1 resulted in G1/G0 arrest in the cell cycle.When OAZ1 or OAZ2 gene was co-transfected with ODC gene into B16-F1 cells,over-expression of GFP-OAZ1 fusion protein,not GFP-OAZ2,demonstrated the ability to significantly decrease the total protein level of ODC.We concluded that over-expression of GFP-OAZ1 or GFP-OAZ2 fusion gene could lead to cell cycle arrest in G1/G0 phase,and GFP-OAZ1 fusion protein could stimulate ODC degradation efficiently,but no such function was found on OAZ2 fusion protein.