摘要
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.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2012年第12期2566-2568,共3页
Chinese Journal of Experimental Surgery