人抗酶抑制因子的克隆与原核表达 - 第三军医大学学报.docVIP

高表达OAZI-1增加黑素瘤B16-F1细胞对多胺类似物CPENSpm的敏感性 （三峡大学分子生物学研究所，湖北宜昌，443002 【摘要】目的：检测高表达OAZI-1的B16-F1细胞对抗癌药物CPENSpm敏感性并探讨其作用机制。MTT法检测细胞增殖；QT-RT-PCR检测细胞内OAZI-1、ODC和OAZ的mRNA表达水平；反相高效液相色谱法检测细胞内多胺含量；化学发光法分析细胞内SMO酶活性。高表达OAZI-1显著增加B16-F1细胞对CPENSpm的敏感性，QT-RT-PCR结果显示，OAZI -1 高表达增加ODC mRNA 但降低OAZ mRNA水平，CPENSpm处理对ODC和OAZ的mRNA水平无进一步影响。 与对照细胞相比，CPENSpm 处理能更显著性地降低OAZI-1高表达细胞中的多胺含量，同时更强地诱导 SMO酶的活性。CPENSpm处理能在更大程度上耗竭OAZI-1高表达的B16-F1细胞中的多胺，由此对该肿瘤细胞显示出更大的细胞毒性。抗酶抑制因子；多胺类似物；黑色素瘤细胞Overexpression OAZI-1 increase the sensitive of the B16 to the polyamine analogues CPENSpm [ABSTRACT] Objective: To detect the influence of OAZI-1 overexpression on the sensitivity B16-F1 cells to polyamine analogues CPENSpm and to assay its mechanism. Method: MTT method was used to detect cell proliferation; QT-RT-PCR was performed to elucidate expression of OAZI-1, ODC, OAZ in mRNA level; polyamine contents in B16-F1 cells were assayed by RP-HPLC, and the chemiluminescience analysis was used to determine SMO enzyme activity. Results: OAZI-1 overexpression increased the sensitive of the B16-F1 to CPENSpm significantly. Increased basic ODC expression and deceased basic OAZ expression in mRNA level were observed in the B16-F1 cells with OAZI-1 overexpression. No further change of ODC and OAZ expression in mRNA level were detected in both the B16-F1 with OAZI-1 overexpression (B16/over) and the control cell (B16/3.1) when treated with CPENSpm for 24h. Comparing with the B16/3.1 control cells, the polyamine contents in B16/over were lower and the SMO active was higher after CPENSpm treatment for 24h. Conclusion: In the B16-F1 cells with OAZI-1 overexpression, CPENSpm can deplete intracellular polyamine more quickly that results in increased sensitivity of B16-F1 cells to the antitumor cytotoxicity of CPENSpm. [Key Words]: ornithine antizyme inhibitor-1; CPENSpm; polyamine; melanoma cell 0 引言 多胺（包括腐胺，精脒和精胺）为肿瘤细胞快速生长所必需，此多胺代谢途径成为抗肿瘤治疗的新靶点。鸟氨酸脱羧酶（Ornithine decarboxylase, ODC）是多胺合成代谢途径中关键的限速酶。ODC催化鸟氨酸脱羧生成腐胺，腐胺继而在精脒合成酶和精胺合成