【作者】 王梦华；

【导师】 李秋明； 郑广瑛；

【作者基本信息】 郑州大学 ， 眼科学， 2004， 硕士

【摘要】 白内障囊外摘除(ECCE)联合人工晶体(IOL)植入术是治疗各种白内障的主要手段，但该手术的主要术后并发症后发障可严重影响患者的视力。在手术后的1个月～5年，约有50％以上的病人出现后囊混浊(posterior capsule opacification，PCO)。而儿童白内障术后PCO的发生率几乎为100％。 目前的研究认为：PCO的形成是由于白内障囊外摘除术后血—房水屏障遭到破坏，导致外源性的细胞因子和炎症介质进入囊袋内，作用于前囊残留的晶状体上皮细胞上，使其发生成纤维细胞样改变，并向后囊不断增殖、迁徙，合成大量的胶原和细胞外基质，这些物质相互混杂，在后囊上形成膜状混浊物。该混浊物可引起后囊皱缩、变形，形成光散射，最终导致视力严重下降。 为了解PCO的形成机制，国外进行了多种体外模拟PCO的培养研究，为体外条件下观察PCO的形成过程和发生机制提供了优越的模型，而国内未见此方面的报道。此次实验我们欲进行牛晶状体后囊的组织培养，体外模拟PCO的形成，观察PCO形成过程中后囊晶状体上皮细胞的增殖分化规律以及血清对体外PCO模型的形成影响。普拉洛芬(Pranoprofen)属于非甾体类抗炎药物，它可明显改善白内障术后及眼前段非感染性炎症患者的主要症状和体征。但关于普拉洛芬对晶状体上皮细胞的抑制实验报道较少，因此，本次实验通过后囊的组织培养，进一步模拟普拉洛芬滴眼液滴眼后4小时在房水中的浓度，观察其对PCO的形成有无抑制作用。郑州大学20()4届硕士学位论文体外模拟后发障的组织培养及药物防治研究 一、实验方法 在无菌条件下，将分离的牛晶状体后囊细胞面朝上平铺于25ml培养瓶底部，分别加入含O、10%、20%胎牛血清的DMEM培养液2.sml，将培养瓶竖直放入37℃、5%coZ的培养箱中，培养液蒸发的水蒸汽可维持晶状体后囊表面湿润，1小时后将培养瓶放平，使囊膜完全浸入培养液中，可见晶状体后囊已于培养瓶的底部牢牢粘附，培养液不会使后囊卷曲、漂浮，再将培养瓶放入培养箱中培养，每3天更换培养液1次，直到后囊晶状体上皮细胞达到融合。培养的各阶段，在倒置显微镜下，用方格图计数板计数后囊晶状体上皮细胞的覆盖率和融合时间，并用Giemsa染色和扫描电镜观察晶状体上皮细胞的形态。同时在含0和2%胎牛血清的培养基中加入普拉洛芬，使其成为体内代谢4小时后的房水内浓度(0.23m岁L)，进行体外培养，观察与对照组后囊上皮细胞融合天数有无差异。 二、实验结果: 赤道部的晶状体上皮细胞向后囊中心部迅速增殖、迁徙，一般约需8一18天可达到融合。细胞的增殖覆盖率与培养液中血清的含量呈正相关，融合时间与血清含量呈负相关(P<0.05)。后囊晶状体上皮细胞发生成纤维细胞样改变，并合成大量的细胞外基质后囊形成明显的混浊和皱缩。培养液中有无血清均可出现后囊皱缩，且血清的含量越高，皱缩形成的数目越多，时间越短。对皱缩区域的Giemsa染色和扫描电镜观察显示:各皱缩纹理排列几乎平行，皱缩边缘有大量的晶状体上皮细胞聚集，且细胞的聚集方向与皱缩纹理的长轴平行。在两种培养状态下，普拉洛芬在体内代谢4小时后的房水内浓度(0 23m留L)对后囊上皮细胞的融合均有明显的抑制作用(P<0.01)。 三、讨论: 1.组织培养条件下的后囊能出现许多与体内PCO相似的改变，如晶状体上皮细胞迅速地在后囊上增殖、纤维化，定向收缩使后囊形成皱缩，并分泌大量的细胞外基质。它是体外模拟PCO形成和研究后发障发生机制的有效方法。 2.血清对体外PCO形成的影响说明:血一房水屏障破坏在PCO形成的早期起到重要作用。后囊无血清组织培养结果显示:晶状体上皮细胞存在“自分泌”机制，PCO的形成是由于“自分泌机制”和“旁分泌机制”共同作用的郑州大学2004届硕士学位论文体外模拟后发障的组织培养及药物防治研究结果。 3.普拉洛芬是一种安全、有效的降低PCO发生率的药物，它可作为白内障术后的常规用药。更多还原

【Abstract】 In recent years, extracapsular cataract extraction (ECCE) and intraocular lens (IOL) implantation play the main role in cataract surgery. Posterior capsule opacification (PCO) , the major complication after ECCE, will affect the paients’ visual acuity seriously. PCO occurs in up to 50% of paients between 1 month and 5 years after surgery., and occurs almost 100% in children.Researchse showed that the formation of PCO is because breakdown of the blood-aqueous barrier after ECCE, which lead to growth factor and inflammatory medium enter capsular bag and act on residual lens epithelial cells on the anterior capsule.The fibroblast-like cells migrate and proliferate rapidly on posterior capsule and secrect a lot of collagen and extracellular matrix. Opaciflcation occurs as a result of the formation of fibrotic membranes on the posterior capsule. Opaciflcation will result in the formation of wrinkle and distortion, which lead to light scattering and visual acuity decrease seriouslyIn order to find out the mechanism of PCO formation, a lot of model forPCO in vitro had been established overseas that provided a good example to observe the process and mechanism of PCO formation ,but the report is seldom in domestic. In this research, tissue culture of lens epithelial cells on the posterior capsule to model posterior capsule opacification (PCO) will be erected, to observe the proliferation of cells and the affection of serum on model for PCO in vitro.the inhibition effects of pranoprofen on PCO were observed. Pranoprofen is a non-steroidal anti-inflammatory drug.In this research, we model pranoprofen whose concentration was like to that has metabolized for 4 hours in aqueous humor (0.23 mg/L) and observe the inhibition of pranoprofen on PCO formation.1. MethedTo spread out the bovine lens posterior capsule with the cell layer upwards on the surface of a 25ml culture flask in sterile condition. DMEM with 0 ,10% and 20% fetal calf serum 2.5ml used as medium were added respectively. The flask was set on ites end in 37 5%CO incubator. The rising water vapour protected the tissue from drying, forming a wet chamber. After 1 hour the flask was laid down,so that the medium covered the epithelium and continue to culture.The medium was changed every 3 days until the confulence of lens epithelial cells on posterior capsule. The cell coverage and confluence time on posterior capsule were recorded with graticule by inverted microscope, and the cell morphology was observed by Giemsa staining and scanning electron-microscope. The lens epithelial cells on posterior capsule were cultured in medium with pranoprofen whose concentration was like to that has metabolized for 4 hours in aqueous humor (0.23 mg/L), and then observed the difference of confluence time with control group.2. ResultThe lens epithelial cells migrate and proliferate rapidly on posterior capsule from equatorial region to the center. Within 8 to 18 days the cells became confluent. The cell coverage was rising with the increase of serum concentration, and the cell confluence time was shortening with that (P < 0.05). The lens epithelial cells on posterior capsule make a fibroblast-like change and secrect a lot of collagen and extracellular matrix. The PCO and wrinkles were formed obviously. The wrinkles presence were not dependent upon serum concentration, but increased in munber and decreased in time with that. The morphology of wrinkles by Giemsa staining and scanning electron-microscope show: the wrinkles almostly paralleled with each other., there were closely packed cells along that, and the direction of packed cells were alinged mainly at right angles to the wrinkles. In two kind of culture condition, Pranoprofen whose concentration was like to that has metabolized for 4 hours in aqueous humor (0.23 mg/L) can suppress the confluence of lens epithelial cells on posterior capsule (P < 0.01).3. Conclusion(1) The posterior capsule in tissue culture shows many of the changes seen in vivo, including rapid cells growth and fibrosis, wrinkles, extracellul更多还原