丝裂原细胞外信号反应激酶阻断剂对缺氧诱导人视网膜色素上皮细胞增殖的影响  

Effect of extracellular signal-regulated kinase of mitogen on cell proliferation of human retinal pigment epithelium stimulated by hypoxia

作　　者：龚琦[1] 袁志兰[1] 于焱[1] 计江东[1] 薛信君[1] 

机构地区：[1]南京医科大学第一附属医院眼科,江苏省南京市210029

出　　处：《中国临床康复》2006年第21期85-87,共3页Chinese Journal of Clinical Rehabilitation

基　　金：国家自然科学基金资助项目(30000207)~~

摘　　要：目的:观察特异性丝裂原细胞外信号反应激酶1(MEK1)阻断剂(PD98059)对缺氧刺激的人视网膜色素上皮细胞增殖的影响,并初步探讨其作用机制。方法:实验于2004-11/2005-11在江苏省人民医院中心实验室完成。①利用体积分数为0.95的N2和0.05的CO2混合气体建立培养的人视网膜色素上皮细胞细胞缺氧模型。②用3种不同浓度(10,50和100μmol/L)的P42/P44MAPK特异性阻断剂PD98059处理视网膜色素上皮细胞细胞。③在缺氧24h后用四甲基偶氮唑盐的方法检测细胞的增殖情况。④用反转录-聚合酶链反应的方法检测周期蛋白DmRNA的表达。⑤收集细胞进行流式细胞仪的检测,判断细胞的周期情况。结果:①缺氧刺激24h后视网膜色素上皮细胞增殖,缺氧组比正常组增加4.71%。②PD98059对视网膜色素上皮细胞细胞增殖有明显的抑制作用,并具有浓度依赖性;与缺氧组相比,缺氧+10mmol/L组、缺氧+50mmol/L组及缺氧+100mmol/L组抑制率分别是5.11%,11.66%和16.36%。③缺氧24h后,细胞周期蛋白DmRNA的浓度明显增加,不同浓度的PD98059对周期蛋白D的mRNA生成有明显的抑制作用,并且呈浓度依赖性,缺氧组与正常组比较周期蛋白D增加26.67%,与缺氧组比较,缺氧+10mmol/L组、缺氧+50mmol/L组及缺氧+100mmol/L组抑制率分别为18.93%,37.72%,57.58%。④缺氧时DNA合成前期细胞明显增多,细胞的增殖减少。结论:P42/P44MAPK信号转导通路可能通过对周期蛋白D的影响抑制缺氧时视网膜色素上皮细胞的增殖。AIM： To investigate the effect of PD98059, the blocking agent of specific mitogen extracellular signal-regulated kinase 1 （ERK1） on the cell proliferation of human retinal pigment epithelium （RPE） stimulated by hypexia, and primarily probe into its mechanism. METHODS： The experiment was conducted in the Central Laboratory of Jiangsu Provincial People＇s Hospital from November 2004 to November 2005.①Hypoxic RPE model that cultured and established with the mixture of N2 （0.95） and CO2 （0.05） were used.②RPE was dealt with P42/P44 MAPK specific blocking agent PD98059 at three different concentration.③ At 24 hours after hypoxia, cell proliferation was assessed by methyl thiazolyl tetrazolium （MTT） colorimetric assay.④The RT-PCR was used to detect the expression of cyclin D mRNA. ⑤ Cells were collected for inspection with flow cytometry, and the cycle of cells was identified. RESULTS： ① At 24 hours after being stimulated by hypoxia, RPE proliferated, which was 4.71% greater in the hypexia group than in the normal control group. ② PD98059 could significantly inhibit the proliferation of RPE and was in a does-dependent manner. Compared with the hypexia group, the inhibition ratio in ＋ 10 mmol/L hypoxia group, ＋50 mmol/L hypoxia group and ＋100 mmol/L hypoxia group was 5.11%, 11.66% and 16.36% respectively. ③At 24 hours after hypoxia, the concentration of cyclin D mRNA significantly increased, and PD98059 of different concentration had significant inhibitory effects on cyclin D mRNA, which was in a dose-dependent manner. Cyclin D in the hypexia group increased 26.7% than in normal group, and compared with the hypoxia group, the inhibition ratio in ＋10 mmol/L hypoxia group, ＋50 mmol/L hypoxia group and ＋100 mmol/L hypoxia group was 18.93%, 37.72% and 57.58% respectively.④Before the synthesis of DNA, the cells were obviously increased in hypexia, whereas the cell proliferation was remarkably decreased. CONCLUSION： The signal transduction pathway of P42/P44 MAPK ma

关 键 词：视网膜 色素上皮 眼/细胞学 丝裂原蛋白激活激酶类 细胞周期蛋白D1 缺氧 细胞增殖 

分 类 号：R318[医药卫生—生物医学工程]