后囊膜混浊的体外上皮-间质转化模型的建立

背景 上皮-间质转化(EMT)是导致后囊膜混浊(PCO)的主要发病机制之一,研究LECs的EMT过程对PCO的防治具有重要意义,但目前缺乏研究EMT的PCO体外细胞模型. 目的 建立新的人晶状体囊外摘出术PCO的体外囊袋培养模型,探讨PCO形成过程中LECs EMT的发生情况.方法 利用健康供体的40只尸眼行体外模拟的白内障摘出术,游离晶状体囊袋后用昆虫针固定于培养皿,置于含体积分数10％胎牛血清的DMEM/F12培养液中培养4周,分别于培养后0、3、7、14和28 d在光学显微镜下观察培养囊袋中LECs的生长情况;收集0、3、7和28 d的囊袋组织制备组织病理学切片,采用苏木精-伊红染色法检测囊袋上LECs的细胞形态;采用免疫组织化学染色法检测EMT标志物α-SMA、E-cadherin和Vimentin蛋白在囊袋上LECs中的表达和定位;采用实时荧光定量PCR和Western blot法检测不同时间点囊袋上LECs中α-SMA、E-cadherin和Vimentin mRNA及其蛋白的表达变化. 结果 未进行培养的囊袋后囊膜上未发现LECs生长,囊袋培养后3d可见后囊膜周边出现LECs并逐渐向中央区增生,培养后7 d LECs完全覆盖后囊膜,略呈铺路石样外观并出现皱褶,此后随着培养时间的延长皱褶的数量增多并伸长,囊袋张力增强.免疫组织化学染色结果显示,随培养时间的延长,囊袋上LECs中E-cadherin的表达强度逐渐下降,而α-SMA和Vimentin的表达强度逐渐增强.实时荧光定量PCR检测表明,培养后0、3、7、14和28 d囊袋上LECs中E-cadherin mRNA的相对表达量分别为3.35±0.13、1.47±0.20、1.13±0.14、1.00±0.85和0.23±0.03,Vimentin mRNA的相对表达量分别为1.00±0.73、1.05±0.14、2.24±0.43、2.84±0.34和8.570±0.40,α-SMA mRNA的相对表达量分别为1.00±0.06、2.68±0.28、4.24±0.05、2.05±0.90和15.30±0.19,总体比较差异均有统计学意义(E-cadherin mRNA:F=23.430,P=0.000;Vimentin mRNA:F=8.915,P=0.002;α-SMA mRNA:F=103.500,P=0.000),其中培养后28 d囊袋上LECs中E-cadherin mRNA的相对表达量最低,而Vimentin mRNA和α-SMA mRNA的相对表达量均最高,差异均有统计学意义(均P＜0.01).Western blot检测结果表明,培养后0、3和28 d囊袋上LECs中E-cadherhin蛋白表达的灰度值分别为1.443±0.017、1.023±0.003和0.568±0.018,Vimentin蛋白表达的灰度值分别为0.565±0.012,1.156±0.007和1.241 ±0.009,α-SMA蛋白表达的灰度值分别为0.195±0.045,0.693±0.036和1.501±0.005,总体比较差异均有统计学意义(E-cadherhin:F=2 787.000,P=0.000:Vimentin:F=4 488.000,P=0.000;α-SMA:F=1 173.000,P=0.000),其中培养后3、28 d LECs中E-cadherhin蛋白表达明显低于0d,而Vimentin和α-SMA蛋白表达明显高于0d,差异有统计学意义(P＜0.05).结论 本研究中的新囊袋模型进行LECs体外培养能模拟体内白内障术后晶状体后囊LECs的EMT自然过程,是探讨PCO发生机制和方法新的体外细胞模型.

In vitro epithelial-mesenchymal transition model for LECs of human posterior capsule opacification

Background Epithelial-mesenchymal transition (EMT) is one of the pathogenesis mechanisms of posterior capule opcification (PCO).Studying EMT is of important significance for the prevention and treatment of PCO.However,EMT model is lack.Objective This study was to establish an in vitro EMT model for the study of human PCO.Methods In vitro mimic cataract enucleation was performed on 40 donor eyes,including anterior capsulorhexis,nucleus hydroexpression,and aspiration of lens fibers.The capsular bag of lens was dissected free during the surgery and pinned flat on a plastic culture dish with DMEM/F12 supplemented containing 10％ fetal bovine serum for 4 weeks.The proliferation of LECs on the capsular bag was observed by phase-contrast and dark-field microscope in 0,3,7,14 and 28 days after culture.The capsular bag tissues were collected in cultured 0,3,7 and 28 days for the preparation of sections and hematoxylin-eosin stain,and the growth and morphology of LECs were examined with optical microscope.The expression and location of α-SMA,E-cadherin and Vimentin were assessed by immunochemistry.The expression levels of α-SMA,E-cadherin and Vimentin mRNA and proteins were detected by using real-time fluorescnce quantitative PCR and Western blot in different time points.Results No LECs were seen on the uncultured capsular bag.LECs appeared in cultured day 3 on the periphery capsular bag and grew toward the center and covered the posterior capsule 7 days after cultured,with a cobblestone-like appearance.Wrinkles occurred and extended gradually along with the enhancement of bag tension.Immunochestry showed that the expression intensity of E-cadherin in the capsular bag gradually weakened,and that of α-SMA or Vimentin was gradually enhanced during the culture duration.The relative expression levels of E-cadherhin mRNA at 0,3,7,14 and 28 days after culture were 3.35±0.13,1.47±0.20,1.13±0.14,1.00±0.85 and 0.23±0.03,and relative expression levels of Vimentin mRNA were 1.00 ± 0.73,1.05 ± 0.14,2.24 ± 0.43,2.84 ± 0.34 and 8.57 ± 0.40,and those of α-SMA mRNA were 1.00 ± 0.06,2.68 ± 0.28,4.24 ± 0.05,2.05 ± 0.90 and 15.30 ± 0.19,showing significant differences among different time points (E-cadherhin mRNA:F =23.430,P =0.000;Vimentin mRNA F =8.915,P =0.002;α-SMA mRNA:F =103.500,P =0.000),with the lowest expression levels in the E-cadherhin mRNA and the highest expression levels in the Vimentin mRNA and α-SMA mRNA at 28 days during the culture period (all at P＜0.01).The gray values of E-cadherin protein expression were 1.443 ± 0.017,1.023 ± 0.003 and 0.568 ± 0.018,and those of Vimentin protein were 0.565±0.012,1.156±0.007 and 1.241±0.009,and those of α-SMA protein were 0.195±0.045,0.693±0.036 and 1.501±0.005 at 0,3 and 28 days,with significnant differences among various time points (E-cadherin:F =2 787.000,P =0.000;Vimentin:F =4 488.000,P =0.000;α-SMA:F =1 173.000,P =0.000).The expression levels were significantly declined in E-cadherhin protein and elevated in Vimentin and α-SMA proteins at 3 and 28 days after culture in comparison with before culture.Conclusions A novel in vitro EMT model of LECs is established in this study.This model can mimic a natural EMT procedure after extracapsular cataract enucleation and therefore is a useful model for the further research of the mechanism and prevention and treatment of PCO.