[1]邱梅园,丁芝祥,廖妙云,等.表没食子儿茶素没食子酸酯对视网膜色素上皮细胞增殖及相关因子表达的影响[J].眼科新进展,2018,38(7):638-642.[doi:10.13389/j.cnki.rao.2018.0150]
 QIU Mei-Yuan,DING Zhi-Xiang,LIAO Miao-Yun,et al.Effects of epigallocatechin gallate on proliferation of retinal pigment epithelium cells and the expression of P21,P27,PDGFR,NF-κB and IκB[J].Recent Advances in Ophthalmology,2018,38(7):638-642.[doi:10.13389/j.cnki.rao.2018.0150]
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表没食子儿茶素没食子酸酯对视网膜色素上皮细胞增殖及相关因子表达的影响/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
38卷
期数:
2018年7期
页码:
638-642
栏目:
实验研究
出版日期:
2018-07-05

文章信息/Info

Title:
Effects of epigallocatechin gallate on proliferation of retinal pigment epithelium cells and the expression of P21,P27,PDGFR,NF-κB and IκB
作者:
邱梅园丁芝祥廖妙云黄岚珍
541001 广西壮族自治区桂林市,桂林医学院附属医院眼科(邱梅园,丁芝祥,廖妙云);541001 广西壮族自治区桂林市,桂林医学院科学实验中心(黄岚珍)
Author(s):
QIU Mei-YuanDING Zhi-XiangLIAO Miao-YunHUANG Lan-Zhen
Ophthalmology,the Affiliated Hospital of Guilin Medical College(QIU Mei-Yuan,DING Zhi-Xiang,LIAO Miao-Yun),Guilin 541001,Guangxi Zhuang Autonomous Region,China;Science Experiment Center of Guilin Medical College(HUANG Lan-Zhen),Guilin 541001,Guangxi Zhuang Autonomous Region,China
关键词:
表没食子儿茶素没食子酸酯人视网膜色素上皮细胞增殖抑制
Keywords:
epigallocatechin gallateretinal pigment epitheliumproliferation inhibition
分类号:
R774
DOI:
10.13389/j.cnki.rao.2018.0150
文献标志码:
A
摘要:
目的 研究表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate,EGCG)对人视网膜色素上皮(retinal pigment epithelium,RPE)细胞增殖及相关因子表达的影响。方法 体外培养人RPE细胞,设阴性对照组,40 mg·L-1、 80 mg·L-1、 160 mg·L-1 EGCG 3个浓度药物处理组。利用MTT检测细胞增殖,流式细胞术检测细胞周期,实时荧光定量PCR检测细胞周期素依赖性激酶抑制因子P21和P27 mRNA的表达,Western blot检测色素上皮衍生因子受体(pigment epithelium derived factor receptor,PDGFR-α)、核因子-κB(nuclear factor-κB,NF-κB)和IκB蛋白的表达情况。结果 MTT实验结果显示,40 mg·L-1、80 mg·L-1、160 mg·L-1 EGCG药物处理组随着作用时间的延长,对人RPE细胞的增殖抑制作用也逐渐增强;随着药物浓度的增加,EGCG的增殖抑制作用也增强(均为P<0.05)。160 mg·L-1 的EGCG 在72 h的细胞增殖抑制率达到81.11%。随着EGCG浓度的增加,培养的人RPE细胞中G1期和G2期细胞比例减少,S期细胞比例增加,80 mg·L-1、160 mg·L-1EGCG药物处理组S期细胞比例与阴性对照组相比增加极显著(均为P<0.05)。EGCG对细胞周期的影响作用具有明显的剂量依赖性。与阴性对照组相比,80 mg·L-1、160 mg·L-1EGCG药物处理组细胞内的P21和P27 mRNA表达均有不同程度的升高(均为P<0.05);40 mg·L-1 EGCG药物处理组与阴性对照组相比,P27 mRNA表达也增高(P=0.015),而P21 mRNA表达增高不明显(P=0.824)。P21和P27 mRNA表达量与EGCG浓度呈明显量效依赖关系(均为P<0.05)。Western blot结果显示,40 mg·L-1、80 mg·L-1、160 mg·L-1 EGCG作用人RPE细胞后,NF-κB蛋白表达随着浓度的增加而减少,各药物处理组与阴性对照组相比差异均有统计学意义(均为P<0.05)。80 mg·L-1、160 mg·L-1 EGCG亦能够抑制人RPE 细胞IκB蛋白和PDGFR-α蛋白表达,与相应阴性对照组相比差异均有统计学意义(均为P<0.05)。EGCG对人RPE细胞内PDGFR-α、NF-κB和IκB的蛋白表达抑制作用呈明显的浓度依赖性(均为P<0.05)。结论 EGCG可以有效地抑制人RPE细胞的增殖,其机制可能与EGCG将人RPE细胞阻滞于S期,上调P21及P27 mRNA表达,下调PDGFR-α、NF-κB和IκB蛋白表达有关。
Abstract:
Objective To study the effect of epigallocatechin-3-gallate (EGCG) on the proliferation and cell cycle of human retinal pigment epithelial cells (hRPE).Methods Human retinal pigment epithelial cells (hRPE) were cultured in vitro and randomly divided into negative control group,40 mg·L-1,80 mg·L-1 and 160 mg·L-1 epigallocatechin gallate (EGCG) treated groups.The cell proliferation was detected by MTT,the cell cycle arrest was detected by flow cytometry,the expression levels of Cyclin-dependent kinase inhibitors P21 and P27 were detected by quantitative real-time PCR (QPCR),and the expression of PDGF NF-κB and IκB protein was detected by Western blot.Results The results of MTT showed that with the increase of EGCG concentration,the growth of hRPE gradually decreased,and EGCG could inhibit significantly the proliferation of hRPE cells.With the treated time went by,the inhibition rates increased,as the drug concentration increased,the inhibition rates were also increasing (both P<0.05).The inhibitory rate of 160 mg·L-1 EGCG on hRPE cells even reached to 81.11% at 72 h,and its inhibitory effect was in a time-dependent and concentration-dependent way.Flow cytometry showed that G1 phase and G2 phase cells gradually decreased,while S phase cells gradually increased with the increase of EGCG concentration.Compared with the negative control group,the proportion of S phase cells in the 80 mg·L-1 and 160 mg·L-1 EGCG treatment groups increased significantly (both P<0.05),the cell cycle results showed that EGCG could block RPE cells in the S phase of the cell cycle.The effect of EGCG on cell cycle was on a dose-dependent pathway.Our cell cycle result showed that EGCG could effectively inhibit the normal cell cycle transition of RPE,allowing cells to accumulate in the S phase,thereby preventing cell mitosis and inhibiting cell proliferation.Compared with the negative control group,the expression of P21 and P27 mRNA in the 80 mg·L-1 and 160 mg·L-1 EGCG-treated groups increased to varying degrees (all P<0.05).Compared with the negative control group,the expression of P27 mRNA was also increased in the 40 mg·L-1 EGCG treatment group (P=0.015),but the expression of P21 mRNA was not significantly increased (P=0.824).The effect of EGCG on the expression of P21 and P27 showed a dose-dependent pathway (all P<0.05).These above results showed that the inhibitory effect of EGCG on cell cycle was related to its up-regulation of cyclin-dependent kinase inhibitor P21 and P27 mRNA expression.Western blot results showed that after 40 mg·L-1,80 mg·L-1 and 160 mg·L-1 EGCG treatment,NF-κB protein expression of human RPE cells was decreased as the EGCG concentration increased,the differences between each drug treatment groups and the negative control group were statistically significant (all P<0.05).And 80 mg·L-1 and 160 mg·L-1 EGCG also inhibited the expression of IκB and PDGF protein in human RPE cells,and the difference was statistically significant compared with the negative control group (both P<0.05).The inhibitory effects of EGCG on the expression of PDGF,NF-κB and IκB in human RPE cells were on an obviously concentration-dependent manner (both P<0.05).Conclusion EGCG can effectively inhibit the proliferation of hRPE cells.The mechanism may be related to EGCG blocking RPE cells in S phase,up-regulating the gene expression of P21 and P27,and inhibiting the protein expressions of PDGF,NF-κB and IκB.

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更新日期/Last Update: 2018-07-12