吲哚美辛对脉络膜黑色素瘤细胞增生活性的影响及作用机制

Indomethacin suppress the proliferation and invasion of human choroidal melanoma cells

目的 观察环氧化酶抑制剂吲哚美辛（IN）对脉络膜黑色素瘤细胞OCM-1增生活性的影响并探讨其作用机制.方法 体外培养OCM-1细胞,采用25、50、100、200、400μmol/L IN对OCM-1细胞进行干预,应用四氮唑化合物（MTT）法检测不同浓度IN对OCM-1细胞增生活性的影响;侵袭实验检测IN对OCM-1细胞侵袭力的影响;逆转录聚合酶链反应（RT-PCR）检测IN对OCM-1细胞内血管内皮生长因子（VEGF）及凋亡抑制因子（survivin）mRNA表达的调节作用;免疫荧光化学检测OCM-1细胞内VEGF、survivin蛋白的定位及表达,酶联免疫吸附测定（ELISA）及蛋白免疫印迹法（Western blot）检测IN对OCM-1细胞内VEGF、survivin蛋白表达的影响.结果 不同浓度IN对OCM-1细胞的增生及侵袭均有明显的抑制作用,其抑制率呈浓度-时间依赖性（MTT：24 h：F=19.642,P＜0.01;48 h：F=136.597,P＜0.01;72 h：F=582.543,P＜0.01;侵袭实验：F=54.225,P＜0.01）.免疫荧光化学检测结果 显示：VEGF、survivin 2种因子在OCM-1细胞内均呈阳性表达且多数表达于细胞胞浆内;RT-PCR检测结果 显示：100、200、400μmol/L IN可抑制OCM-1细胞内survivin mRNA的表达（F=16.679,P＜0.01）,但对细胞内VEGF mRNA的表达无抑制作用.ELISA法测出未用药组、100、400 μmol/L IN作用OCM-1细胞24 h后,细胞内survivin的浓度分别为（787.33±47.37）、（257.00±26.21）、（123.33±8.02）pg/ml;Western blot进一步验证了IN可抑制OCM-1细胞内survivin蛋白的表达,但对VEGF的表达无抑制作用.结论 IN能够抑制OCM-1细胞的增生及侵袭,其机制可能与其下调OCM-1细胞内survivin因子的表达有关.

Objective To observe the influence of the indomethacin on the proliferative and invasive activity of OCM-1 human choroidal melanoma cells. Methods OCM-1 cells were cultured with different concentrations of indomethacin （25, 50, 100, 200, 400 μmol/L ）, and their proliferation were assessed by methyl thiazolyl tetrazolium（MTT）, invasive behaviors were examined by cell invasion assays, expression of survivin and VEGF were evaluated by reverse transcriptase polymerase chain reaction （RT-PCR）,immunofluorescence staining, ELISA and western blot analysis. Result All concentrations of indomethacin in this study can inhibit the proliferation and invasion of OCM-1 cells in a time and dosage-dependant manner （MTT/24 h： F= 19. 642, P〈0.01;MTT/48 h： F= 136. 597, P〈0. 01;MTT/72 h： F= 582. 543, P〈 0. 01;invasion assays： F= 54. 225, P〈 0. 01）. Immunofluorescence staining indicated that survivin and VEGF mainly expressed in the cytoplasm of OCM-1 cells. Survivin mRNA in OCM-1 cells was inhibited by 100,200, 400 μmol/L indomethacin （F= 16. 679, P〈0.01）. The concentrations of survivin were （787.3±47.37）, （257.0± 26.21）, （123. 3± 8.02） pg/ml in control group and 100, 400 μmol/L indomethacin groups, respectively. Survivin expression was also significantly down-regulated in indomethacin-treated cells by Western blot analysis. Indomethacin had no effects on VEGF expression in OCM-1 cells. Conclusions Indomethacin can inhibit proliferation and invasion of OCM-1 cells in vitro, down-regulated expression of survivin may be the mechanism.