后囊膜混浊的体外上皮-间质转化模型的建立  

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

作　　者：杨宝霞[1] 王晔[2] 赵晓雯[2] 刘廷[2] 黄钰森[2] 

机构地区：[1]济南大学山东省医学科学院医学与生命科学学院,250355 [2]山东省医学科学院山东省眼科研究所山东省眼科学重点实验室——省部共建国家重点实验室培育基地,青岛266071

出　　处：《中华实验眼科杂志》2016年第3期210-217,共8页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目（81370990、81370996）;山东省自然科学基金项目（ZR2012HM009、BS2012YY030）

摘　　要：背景上皮一间质转化（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LECsEMT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28d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28d的囊袋组织制备组织病理学切片，采用苏木精一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OL—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Westernblot法检测不同时间点囊袋上LECs中a-SMA、E—cadherin和VimentinmRNA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7dLECs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a-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28d囊袋上LECs中E—cadherinmRNA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，VimentinmRNA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，d—SMAmRNA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—cBackground Epithelial-mesenchymal transition （EMT） is one of the pathogenesis mechanismsof 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 fiat 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 a-SMA,E-cadherin and Vimentin were assessed by immunochemistry. The expression levels of a-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 a-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

关 键 词：上皮-间质转化 晶状体上皮细胞 后发性白内障 白内障摘出/不良反应 晶状体囊 细 胞培养 人 囊袋模型 

分 类 号：R-332[医药卫生] R776