白藜芦醇诱导人脉络膜恶性黑色素瘤细胞凋亡及其机制研究

Effects of resveratrol on cell apoptosis of the human choroidal melanoma Mum2c cells

目的探讨白藜芦醇（Res）诱导人脉络膜恶性黑色素瘤细胞Mum2c凋亡及其机制。方法应用不同浓度的Res（0、4、8、16、32、48mg／L）处理体外培养Mum2c细胞，24、48、72h后，采用细胞计数试剂盒（CCK-8）法检测细胞增殖。应用不同浓度的Res（32、40、48mg／L）处理Mum2c细胞，24h后采用流式细胞仪膜联蛋白V-异硫氰酸荧光素（AnnexinV，FITC）／碘化丙锭（PI）双标法检测细胞凋亡，线粒体膜电位JC-1荧光检测观察细胞凋亡，检测半胱氨酰天冬氨酸特异性蛋白酶（Caspase）-3活性，免疫细胞化学法检测B淋巴细胞／白血病-2（bcl-2）表达。结果CCK一8检测结果显示16mg／L以上浓度的Res作用24、48、72h后Mum2c增殖与对照组比较差异有统计学意义（P〈0．05）。AnnexinV—FITC／PI染色流式细胞仪检测显示。Res（32、40、48mg／L）作用组凋亡率[（7．00±0．98）％、（11．87±0．60）％、（15．39±1．04）％]，与阴性对照组[（2．02±0．84）％]比较差异有统计学意义（P〈0．05）。jc-1荧光显微镜下观察，可见随浓度增加Res作用组低红染色细胞比例[（7．42±0．53）％、（13．12±0．83）％、（15．57±0．53）％]逐渐增加，与阴性对照组[（3．57±0．53）％]比较差异有统计学意义（P〈0．05）。各Res作用组Caspase-3的吸光度（A）值（0．246±0．015、0．566±0．027、0．778±0．018），与阴性对照组（0．151±0．010）比较差异有统计学意义（P〈0．05）。bcl-2阳性细胞百分率[（49．60±1．51）％、（38．00±1．26）％、（26．67±1．21）％]，与阴性对照组[（61．33±1．21）％]比较差异有统计学意义（P〈0．05）。结论Res能够诱导人脉络膜恶性黑色素瘤Mum2c细胞凋亡，其作用机制与线粒体膜电位被破坏，Caspase-3活性增强，bel-2表达水平降低有关。

Objective To investigate the effect of resveratrol on cell apoptosis of the human cho- roidal malignant melanoma Mum2c cells. Methods Mmn2c cells were cultured in RPMI 1640 medium supplemented with Resveatrol （Res） at concentration of 0, 4, 8, 16, 32, 48 mg/L respectively. After in- cubation for 24, 48, 72 h, cell viability and proliferation was determined by cell counting kit-8 （ CCK-8 ） assay. After.incubation for 24h with Res at conceration of 32, 40, 48 mg/L respectively, the apoptosis was detected by both flow cytomeu：~ （FCM） with Annexin V-FITC/PI staining and JC-1 miochondrial member- ane potential fluorescence. The levels of B lymphocytes/leukemia-2 （bcl-2） protein expression was deter- mined by cellular immunohistochemistry ; the activation of cysteinyl aspartate-specific protease （Caspase） -3 was determined by enzyme linked immunosorbent assay （ELISA）. Results By Res 16 mg/L and above concentration on Mum2c for 24, 48, 72 h, the A absorbance of Mum2c cells was significant different from contol group. After incubation with Res at concentration of 32, 40, 48 mg/L for 24 h, the apoptosis rate [ （7. 00 ±0. 98）%, （11.87 ±0. 60）%, （ 15.39± 1.04）% ] was increased significantly compared with control group （2. 02 ± 0. 84 ） （P 〈 0. 05）. After incubation with Res at concentration of 32, 40, 48 mg/L for 24 h, the percentage of positive cell of expressing bcl-2 [ （49.60 ± 1.51 ）%, （38. 00 ± 1.26）%, （26. 67 ± 1.21 ） % ] was decreased significantly compared with control group [ （61.33 ± 1. 21 ） % 1 （ P 〈 0.05 ）. The activation of Caspase-3 was increased significantly compared with control group （ P 〈 0. 05）. Conclusion Res can inhibit Mum2c proliferation and induce apoptosis, its mechanism ,nay be related with disrupting mitochondrial membrane potential, downregulating the expression of bcl-2 and increasing the activation of Caspase-3.