Gotoh Norihito, Perdue Nikole R, Matsushima Hiroyuki, Sage E Helene, Yan Qi, Clark John I
Department of Biological Structure, University of Washington School of Medicine, Seattle, Washington 98195-7420, USA.
Invest Ophthalmol Vis Sci. 2007 Oct;48(10):4679-87. doi: 10.1167/iovs.07-0091.
This report presents a novel model for studies of extracellular matrix (ECM) in posterior capsular opacification (PCO) in vitro. Lens epithelial cells (LEC) were cultured with an intraocular lens (IOL) on a surface of type IV collagen in an evaluation of the importance of the ECM-cell interaction in formation of PCO. Abnormal migration, proliferation, and expression of proteins associated with the epithelial-to-mesenchymal transition (EMT) that characterizes PCO were observed in the presence and absence of the matricellular protein SPARC (secreted protein, acidic and rich in cysteine), which regulates matrix-cell interactions.
The model for PCO in vitro consisted of an IOL placed on a membrane coated with collagen IV, a major constituent of the lens capsule. LECs from the lenses of wild-type (WT) and SPARC-null (SP-null) mice were cultured in the presence or absence of 10 ng/mL TGF-beta2 and 20 mug/mL recombinant human SPARC (rhSP) for up to 6 days. The migration of LECs was quantified. Labeling with BrdU and the measurement of DNA synthesis were assays for cell proliferation. Expression of the EMT markers, collagen type I, fibronectin, and alpha-smooth muscle actin were assessed using immunocytochemistry or Western immunoblots.
LEC migration, proliferation, and the synthesis of EMT markers were enhanced in SP-null compared with WT LECs. TGF-beta2 inhibited the migration and proliferation of both WT and SP-null LECs in the presence of rhSP. TGF-beta2 increased the production of collagen type I, fibronectin, and alpha-SMA. The responses of SP-null LECs were rescued by the addition of recombinant human (rh)SP.
A simple IOL culture system was useful for the evaluation of the effects of SPARC and TGF-beta2 on PCO in vitro. The action of TGF-beta2 on LEC migration and proliferation is influenced by SPARC, a regulator of matrix-cell interactions. The results indicate a functional intersection between pathways activated by TGF-beta2 and SPARC in the formation of PCO.
本报告提出了一种用于体外研究后囊膜混浊（PCO）中细胞外基质（ECM）的新模型。将晶状体上皮细胞（LEC）与人工晶状体（IOL）在IV型胶原表面进行培养，以评估ECM-细胞相互作用在PCO形成中的重要性。在存在和不存在调节基质-细胞相互作用的基质细胞蛋白SPARC（分泌蛋白，酸性且富含半胱氨酸）的情况下，观察到与PCO特征性的上皮-间充质转化（EMT）相关的异常迁移、增殖以及蛋白质表达。
体外PCO模型由放置在涂有IV型胶原（晶状体囊膜的主要成分）的膜上的IOL组成。来自野生型（WT）和SPARC基因敲除（SP-敲除）小鼠晶状体的LEC在存在或不存在10 ng/mL转化生长因子-β2（TGF-β2）和20 μg/mL重组人SPARC（rhSP）的情况下培养长达6天。对LEC的迁移进行定量。用溴脱氧尿苷（BrdU）标记和DNA合成测量作为细胞增殖的检测方法。使用免疫细胞化学或Western免疫印迹评估EMT标志物I型胶原、纤连蛋白和α-平滑肌肌动蛋白的表达。
与WT LEC相比，SP-敲除的LEC迁移、增殖以及EMT标志物的合成增强。在rhSP存在的情况下，TGF-β2抑制WT和SP-敲除的LEC的迁移和增殖。TGF-β2增加I型胶原、纤连蛋白和α-SMA的产生。添加重组人（rh）SP可挽救SP-敲除LEC的反应。
一个简单的IOL培养系统可用于体外评估SPARC和TGF-β2对PCO的影响。TGF-β2对LEC迁移和增殖的作用受基质-细胞相互作用的调节因子SPARC影响。结果表明在PCO形成过程中，TGF-β2和SPARC激活的信号通路之间存在功能交叉。
