摘要
背景姜黄素是从姜的根茎中提取的物质，可抑制多种内皮细胞和上皮细胞的增生，但其抑制视网膜色素上皮（RPE）细胞增生的作用及机制鲜见报道。目的研究姜黄素对体外培养的人RPE细胞增生的抑制作用及机制。方法第5代人RPE细胞株接种于96孔板，分别加入5、10、15、20mg／L姜黄素分别作用24、48和72h，未加入姜黄素的作为对照，采用水溶性四氮唑-1（WST-1）检测各组人RPE细胞的增生情况和细胞活性（A值）；采用流式细胞术检测姜黄素作用后RPE细胞的凋亡率、坏死率和不同细胞周期的细胞比例；透射电子显微镜（TEM）观察细胞超微结构的变化；采用Westernblot法检测RPE细胞中促凋亡因子p53、p21WAF1/CIPI和增生细胞核抗原（PCNA）蛋白的表达。结果WST-1检测显示，随着姜黄素质量浓度的增加，人RPE细胞的』4值逐渐降低，差异有统计学意义（Fm质量浓度=96．55，P=0．00）；随着姜黄素作用时间的延长A值逐渐增加，差异有统计学意义（FH目=4634．28，P=0．00）。流式细胞术检测表明，15mg／L姜黄素作用48h早期凋亡细胞率为（13．37±1．26）％，明显高于对照组的（7．03±0．37）％，差异有统计学意义（t=8．33，P=0．00），姜黄素作用72h，早期凋亡细胞率及中晚期凋亡细胞率分别为（15．97-＋0．16）％和（0．26-＋0．03）％，明显高于对照组的（7．29±0．37）％和（O．14±0．02）％，差异均有统计学意义（t=37．80、7．44，均P=0．00）。15mg／L姜黄素作用48h后人RPE细胞处于G。／G，期细胞比例为（57．17±1．17）％，明显低于对照组的（67．73±1．10）％，差异有统计学意义（t=11．40，JP=0．00）。姜黄素作用后人RPE细胞可见线粒体肿胀和空泡样变性。Westernblot检测显示，15mg／L姜黄素作用24、48、72h后p53和p21WAF1/CIPI蛋白相对表达值均明显高于对照组，差异均有统计学意义（均P〈0．05），而15mg／L姜黄素作用24、48、72hPCNA蛋白的相对表达水平明显低于对照组，差异均有统计学意义（均P〈0．05）。结论姜黄素可有效抑制体外培养的人RPE细胞增生，其抑制作用呈剂量和时间依赖性，其作用机制可能与p53信号通路有关。
Background Curcumin derives from the rhizome of curcuma longa. It has proven to have an antiproliferative effect in previous studies on vast majority of endothelial and epithelial cells,however,the study of its inhibiting effect on the proliferation of retinal pigment epithelial （RPE） ceils and underlying mechanism is rare. Objective Aim of this study was to investigate the potential inhibitory effect of cureumin on the proliferation of cultured human RPE cells in vitro and its possible mechanism. Methods Human RPE cells harvested by trpsin- EDTA were suspended in DMEM/F12 medium with serial dilutions of eureumin （5,10,15,20 rag/L） ,and the human RPE cells cultured by DMEM/F12 without curcumin were used as control. The proliferation value of human RPE ceils （A value） was measured by water-soluble tetrazole-1 （WST-1） assay, the optimized dose of antiproliferation of curcumin was determined and applied for further experimental process. Apoptosis and cell cycle of human RPE cells were detected by flow cytometrie analysis at 48 hours and 72 hours after curcumin treatment. The uhrastrueture profile of the cells were examined by transmission electron microscopy （TEM）. Western blot analysis was performed to measure the relative expressing level of the pro-apoptotic factors p53, p21WArl/clvl and proliferating cell nuclear antigen （PCNA） in the cells,respectively. Factorial design of two factor analysis of variance of SPSS 17.0 software was used to compare the difference of A values of the cells among the various groups and time points,and independent-sample t test was used to eompare the differences of apoptosis rate and eell ratio in different cycles between curcumin group andcontrol group. Results WST-1 assay showed that the A value was gradually reduced with the increase of curcumin dose （ F . = 96.55, P = 0. 00 ） , and gradually increased with the lapse of time （ Fm, = 4634.28, P = 0.00）. The early apoptotic rate of the cells was （13.37 ± 1.26） % in the curcumin group 48 hours after treated by 15 mg/L curcumin, and that of the control group was （ 7.03 ±0.37 ） % , with a significant difference between them （ t = 8.33, P=0.00）. In 72 hours after treated by 15 mg/L curcumin,the early and middle-late apoptotic rates of the cells were （15.97±0. 16） % and （0.26±0.03） % , which were significantly higher than those of the control group （7.29 ± 0.37） % and （0.14-±0.02） % （t=37.80,P=O. OO;t=7.44,P=O. OO）.The cell ratio of G0/G1 phase in the curcumin group was （57. 17±1. 17）% 48 hours after treated by 15 mg/L curcumin,and that in the control group was （67.73-± 1. 10 ） % , showing a significant difference （ t = 11.40, P = 0.00 ）. Mitochondrial swelling and vacuolar degeneration were seen in the cells after treated by 15 mg/L curcumin. The relative expression levels of p53 and p21wsvl/cIP1 protein in the ceils were higher in the curcumin group than those of the control group at 24,48 and 72 hours （ all at P〈0.05 ） ,but the expression levels of PCNA protein were lower in the curcumin group than those of the control group in various time points （all at P 〈 0.05 ）. Conclusions Curcumin can effectively inhibit the proliferation of human pigment epithelial cells in a dose- and time-dependent manner. P53 pathway may participate in anti-proliferating process.
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2013年第12期1110-1116,共7页
Chinese Journal Of Experimental Ophthalmology
基金
江西省自然科学基金项目（2007GZY1285）
关键词
姜黄素
视网膜色素上皮细胞
凋亡
Curcumin
Retinal pigment epithelial cell
Apoptosis
