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目的:研究鸟氨酸脱羧酶抗酶抑制因子-1(ornithine decarboxylase antizyme inhibitor-1,OAZI-1)高表达对小鼠黑素瘤B16-F1细胞增殖的影响。方法:采用巢氏PCR法从小鼠肝癌H22细胞cDNA中克隆出小鼠OAZI-1基因,并构建重组质粒pcDNA3.1(+)/OAZI-1。采用脂质体转染法将重组质粒转染入B16-F1细胞后,Western印迹法和实时荧光定量PCR法鉴定高表达OAZI-1的B16-F1细胞株。MTT法和FCM检测OAZI-1高表达对B16-F1细胞增殖和细胞周期的影响。反相高效液相色谱检测OAZI-1高表达对细胞内多胺水平的影响。化学发光法检测细胞内精胺氧化酶(spermine oxidase,SMO)的活性。结果:成功获得高表达OAZI-1的B16-F1细胞克隆株B16/over,该细胞中OAZI-1在mRNA水平的表达量是转染空载体质粒对照组细胞B16/3.1的3.6倍。B16/over细胞中,鸟氨酸脱羧酶抗酶(ornithine decarboxylase antizyme,OAZ)的mRNA表达水平降低,鸟氨酸脱羧酶(ornithine decarboxylase,ODC)的mRNA表达水平升高。高表达OAZI-1能促进B16-F1细胞增殖,并影响细胞周期,G0/G1期细胞减少而S期和G2/M期细胞增加。B16/over细胞中腐胺的含量明显增加,精脒和精胺的含量减少,而且SMO活性升高。结论:OAZI-1高表达可能通过增加细胞内腐胺含量,促进黑素瘤细胞增殖,提示该抑制蛋白可能成为黑素瘤治疗的一个潜在靶点。
OBJECTIVE: To study the effect of ornithine decarboxylase antizyme inhibitor-1 (OAZI-1) on the proliferation of mouse melanoma B16-F1 cells. Methods: The mouse OAZI-1 gene was cloned from cDNA of mouse H22 cells by nested PCR and the recombinant plasmid pcDNA3.1 (+) / OAZI-1 was constructed. The recombinant plasmids were transfected into B16-F1 cells by lipofection method. The B16-F1 cell lines overexpressing OAZI-1 were identified by Western blotting and real-time fluorescence quantitative PCR. The effects of OAZI-1 overexpression on the proliferation and cell cycle of B16-F1 cells were detected by MTT and FCM. Effect of OAZI-1 overexpression on the level of intracellular polyamines by reverse phase high performance liquid chromatography. Chemiluminescence was used to detect intracellular spermine oxidase (SMO) activity. Results: The B16-F1 cell clone B16 / over which overexpressed OAZI-1 was successfully obtained. The expression level of OAZI-1 at the mRNA level was 3.6 times higher than that of B16 / 3.1 transfected plasmid vector. In B16 / over cells, mRNA expression of ornithine decarboxylase antizyme (OAZ) decreased and mRNA expression of ornithine decarboxylase (ODC) increased. High expression of OAZI-1 can promote the proliferation of B16-F1 cells and affect the cell cycle, G0 / G1 phase cells decreased and S and G2 / M phase cells increased. The content of putrescine in B16 / over cells increased significantly, the content of spermidine and spermine decreased, and the activity of SMO increased. CONCLUSION: OAZI-1 overexpression may promote the proliferation of melanoma cells by increasing intracellular putrescine content, suggesting that this suppressor protein may be a potential target for the treatment of melanoma.