囊袋法兔晶状体上皮细胞体外培养及超微结构观察

Study of rabbit lens epithelial cell's survival and ultrastructure on the rabbit capsular bag in vitro

目的建立一种囊袋模型,研究兔晶状体囊外摘除术(ECCE)后残余的晶状体上皮细胞(LECs)在体外增殖、移行、化生的情况。方法用18只兔眼,模拟白内障手术,体外环形撕囊,水分离,去除核与皮质,游离晶状体囊袋,并固定于硅胶环上,置含10%胎牛血清的DMEM营养液中培养3周。相差显微镜观察不同培养时期后囊上LECs增殖、移行情况并行组织病理学、透射电镜检查及平滑肌肌动蛋白(α-SMA)免疫组织化学染色。结果经过2～3天的潜伏期,后囊周边部开始出现LECs,细胞增殖速度较快,约6～8天可完全覆盖后囊。培养后期,后囊上可出现双层甚至多层细胞,囊膜可见明显皱褶,组织学见均质红染的后囊膜上被覆一层或多层排列紧密﹑核深染、胞浆丰富的LECs。随培养时间延长,皱褶的数量和长度逐渐增多,囊袋光散增加,后囊张力亦明显增强。透射电镜下LECs排列紧密,周界清晰,胞浆内可见丰富的线粒体及内质网等细胞器。培养1周方可见后囊上LECsα-SMA表达,呈散在胞浆棕色染色,α-SMA表达具有明显时间相关性。结论应用这种囊袋模型进行LECs体外培养,较为真实的表现了体内ECCE术后多种变化,有助于更深一步探讨后囊膜混浊的发生机制,并为筛选防治后发性白内障的有效药物,提供了有价值的实验手段。

OBJECTIVE To study the proliferation, migration and metaplasm of residual lens epithelial ceils （LECs）in rabbit after extraeapsular cataract extraction （ECCE）based on the rabbit capsular bag model in vitro. METHODS Same cataract surgery was performed on 18 rabbit lens. The capsular bags were isolated and pinned to sterile non-toxic silicone rings on a Petri dish. They were monitored for 3 weeks by phase-contrast microscopy, after which they were performed for light microscopy and transmission electron microscopy （TEM）. The expression of a-smooth muscle actin （ct-SMA）in LECs on capsular bags was detected with SP immunohistochemical methods. RESULTS Growth proceeded rapidly so that the pos- terior capsule was totally covered by a confluent monolayer of cells at 6-8 days. Under TEM, the ceils were tightly arrayed with clear deformity and undefined intracellular boundary and structure. The expression of α-SMA in LECs was positive after 1 week in culture,and the positive α-SMA expression was obviously increased as time progressed. CONCLUSIONS This model exhibited many characteristics of the lens capsule in vitro after extracapsular surgery and might usefully prove in further elucidating the cellular mechanisms of posterior capsule opacification and developing strategies for inhibiting cell growth with this system.