Background Retinal retinoic acid (RA) plays an important role in the formation of the lensinduced myopia.However,it is not clear how RA transfer the myopic signal to choroid throughout the retinal pigment epithelium (RPE) barrier.Objective The aim of this study was to investigate the effect of all-trans retinoic acid (atRA) on the barrier of RPE in lens-induced myopic eye of guinea pig.Methods Thirty left eyes of 30 21-dayold clean guinea pigs were randomized into normal control group and the model group.The models of out of focus were induced by covering of-6.00 D concave lens on the left eyes for 15 days.Radius of corneal curvature was measured using corneal curvimeter,and diopeter of the guinea pig was examined by mydriatic optometry.The length of ocular axis was detected by A-sonography.The animals were sacrificed and the retinas of the left eyes were isolated for the culture and passage of RPE cells.The third generation of cells were incubated Millcell-PET microporous film,and atRA at the concentration of 1 × 10-6,1 × 10-7,1 × 10-8 and 1 × 10-9 mol/L was added to the micropore respectively for 12 hours,and the micropores with equal-solvent served as negative control group.Methyl thiazolyl tetrazolium (MTT)colorimetric method was used to detect the survival rate of the cells.Subsequently,the transepithelial electrical resistance (TER) of the monolayer cells was determined with CN10-EVOM2 resistance measuring meter.The vesicular transport change of RPE membrane in different groups was evaluated by FM1-43 fluorescence staining.Results The mean diopter was (-2.20±0.95) D in the models,and that in the controls was (+ 1.15 ±0.30) D,with a significant difference between them (t =14.57,P＜ 0.01).The axial length was (8.24 ± 0.09) mm in the models and it was significantly longer than (7.81±0.05) mm in the controls (t=17.20,P＜0.01).RPE cells grew well to form a monolayer in Millcell culture pool after one week.After 24 hours of the atRA treatment,the survival rate of RPE cells reduced gradually with the increase of atRA concentration with the highest rate in the 1 × 10-9 mol/L atRA group (93.3 ％) and followed by the 1 × 10-8 mol/L atRA group (88.2％).More than half of the cells dead in the 1 × 10-6mol/L and 1 × 10-7mol/L atRA groups (53.8％ and 47.1％).Significant differences in the TER value and fluorescence staining intensity of the cells were seen among the various groups (F =43.89,P =0.00 ; F =26.13,P =0.00),with the maximal values in the 1 × 10-8mol/L atRA group.The FM1-43 fluorescence located on the cellular membrane and cytoplasm.Conclusions AtRA can increase the functional state of tight junction and vesicular transport,which regulated the RPE cell barrier in the guinea pig.%背景 视网膜视黄酸在离焦性近视形成中起重要作用,但目前尚不清楚其如何通过视网膜色素上皮(RPE)屏障向脉络膜传递近视信号. 目的 研究全反式视黄酸(atRA)对离焦性近视眼RPE屏障功能的影响.方法 21日龄清洁级三色豚鼠30只,用随机数字表法分为正常对照组和离焦组,-6 D凹透镜缝合于离焦组豚鼠左眼15d以制备离焦性近视模型,每天光照与黑暗周期为12 h/12 h.用角膜曲率计测量各组豚鼠的角膜曲率半径,行复方托吡卡胺滴眼液扩瞳检影验光以测量豚鼠眼球屈光度,应用A型超声法测量各组豚鼠的眼轴长度.于造模15d时处死动物并分离视网膜,体外培养RPE细胞并传代,将第3代RPE细胞用于实验,制备视网膜铺片,接种于Millcell-PET微孔滤膜插入式培养池,分别加入1×10-6、1×10-7 、1×10-8和1×10-9 mol/L atRA培养RPE细胞24 h,以加入等体积atRA溶剂的培养孔作为阴性对照,以完全培养基加MTT溶液的无细胞孔作为空白对照.采用MTT比色法检测各组RPE细胞的存活率,用CN10-EVOM2型电阻测量仪测定单层细胞跨上皮电阻(TER),计算RPE细胞的TER值,采用FM1-43型荧光染色技术检测得出RPE细胞FM1-43阳性荧光染色的相对强度值,评价RPE细胞膜泡运输变化. 结果 豚鼠模型眼屈光度为(-2.20±0.95)D,对照组为(+1.15±0.30)D,差异有统计学意义(t=14.57,P＜0.01);豚鼠模型眼的眼轴长度为(8.24±0.09) mm,明显长于对照组的(7.81±0.05) mm,差异有统计学意义(t=17.20,P＜0.01).原代培养豚鼠近视眼的RPE细胞,取第3代细胞用于实验.RPE细胞接种于Millcell培养池1周后细胞长满滤膜,呈单层生长.加入atRA干预24 h,RPE细胞的存活率随药物浓度的升高而逐渐降低,1×10-9 mol/L atRA组RPE细胞存活率为93.3％,1×10-8mol/L atRA组为88.2％,1×10-6mol/L atRA组和1×10-7 mol/L atRA组的RPE细胞大量死亡.不同浓度atRA组RPE细胞的TER值和RPE细胞FM143阳性荧光染色的相对强度值的总体比较差异均有统计学意义(F=43.89,P=0.00;F=26.13,P=0.00),其中1×10-8mol/L atRA组RPE细胞的TER值为(107.32±9.58) Ω/cm2,荧光染色强度为55.29±9.79,均明显高于1×10-6、1×10-7、1×10-9mol/LatRA组,差异均有统计学意义(P＜0.05),FM143荧光染色部位主要在RPE细胞的细胞膜和细胞质. 结论 AtRA能够通过增加豚鼠近视眼RPE的紧密连接功能及膜泡运输改变RPE细胞的屏障功能.
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