高糖条件下人视网膜色素上皮细胞的上皮-间质转化

Epithelial-mesenchymal transition of human retinal pigment epithelial cells under the high glucose condition in vitro

背景 研究证实,有多种细胞参与增生性玻璃体视网膜病变（PVR）的发生和发展过程,其中RPE细胞的上皮-间质转化（EMT）可能与PVR有关,其主要生物学行为是RPE细胞的增生和迁移.已证实高血糖是糖尿病视网膜病变（DR）发病的主要原因,而高糖条件下RPE细胞是否发生EMT鲜有报道. 目的 建立体外高糖细胞培养模型,探讨高糖对人RPE的迁移能力及EMT的影响.方法 用含质量分数10％胎牛血清的DMEM/F12培养基对人RPE细胞D407细胞株进行体外培养和传代,取6～8代细胞用于实验.依据培养液中血糖浓度的不同将细胞分为3个组,正常对照组培养基中葡萄糖终浓度为5.5 mmol/L,高糖培养组葡萄糖终浓度为60.0 mmol/L,用含5.5 mmol/L葡萄糖和甘露醇的DMEM培养基培养细胞作为高渗对照组.采用细胞划痕试验法检测划痕后0、24、48和72 h时3个组RPE细胞的迁移率,用实时荧光定量PCR（real-time PCR）检测高糖培养组在培养不同时间点RPE细胞间质化标志物闭锁小带蛋白-1（ZO-1）和α-平滑肌肌动蛋白（α-SMA）mRNA在细胞中的相对表达水平.结果 正常对照组培养的RPE细胞呈多角形,核仁清晰,排列致密;高糖培养组随着培养时间的延长,RPE细胞逐渐变大、变长,细胞结构不清,排列紊乱;高渗对照组细胞结构接近正常对照组.划痕试验显示,高糖培养组在划痕后48 h可见细胞迁移,划痕后72 h划痕基本消失,而正常对照组和高渗对照组划痕仍然存在.划痕后各时间点高糖培养组细胞迁移率明显高于正常对照组和高渗对照组,组间和各时间点的差异均有统计学意义（F分组=328.600,P=0.000;F时间=773.270,P=0.000）.RT-PCR结果显示,与正常对照组相比,高糖培养后48 h、72 h组细胞中ZO-1 mRNA的表达水平明显低于正常对照组,而α-SMA mRNA的表达水平明显高于正常对照组,差异均有统计学意义（均P＜0.05）.结论 高糖条件下RPE细胞的迁移能力增强,发生EMT改变,可能参与增生性糖尿病视网膜病变（PDR）的发生.

Background Several types of cells participate in the formation of proliferative membrane in proliferative retinopathy （PVR）,and the proliferation,migration and epithelial-mesenchymal transition （EMT） of retinal pigment epithelium （RPE） cells play an important role.Many studies have confirmed high blood glucose is the basic pathogenesis of diabetic retinopathy （DR）.However,whether EMT could be induced in RPE cells under the high glucose condition has not been reported.Objective This study was to investigate the effects of high glucose on the migration and EMT of RPE cells in high glucose culture model in vitro.Methods Human RPE cell line D407 were cultured and passaged in DMEM/F12 medium with 10% fetal bovine serum,and 6-8 generations of cells were used in experiment.The cells were divided into 3 groups based on different glucose concentrations in medium.The glucose at the final concentration 5.5 mmol/L or 60.0 mmol/L was respectively used in the normal control group or high glucose group,and the DMEM with 5.5 mmol/L glucose and mannitol was used in the hypertonic control group.The migration rate of the cells were detected 0,24,48 and 72 hours after scratching by wound-scratch test.Real-time PCR was used to detect the relative expressions of zonula occludens-1 （ZO-1） and α-smooth muscle actin （α-SMA） in the cells.Results Cultured cells showed a polygon shape with the clear nucleolus and dense arrangement in the normal control group and the hypertonic control group,but the cells were larger and elongated with the lapse of culture time with the indistinct structure and loose arrangement.At 48 hours after scratching,migrating cells were seen in the scratching area,and the scratching area disappeared at 72 hours after scratching in the high glucose group,but the scratching area still was existed in the normal control group or hypertonic control group.The migrating rate of the cells was higher in the high glucose group than that in the normal control group or hypertonic control group,showing total differences among 3 groups and various time points （Fgroup =328.600,P =0.000 ; Ftime =773.270,P=0.000）.Compared with the normal control group,the expression level of ZO-1 mRNA was significantly lower,and α-SMA mRNA level was higher 48 hours and 72 hours in the high glucose group than those in the normal control group （all at P〈0.05）.Conclusions High glucose induce the migration and EMT of RPE cells in vitro,which may be associated with the pathogenesis of proliferative diabetic retinopathy.