眼视光学杂志 2000年第4期第2卷 专稿

作者：胡诞宁　Steven A.McCormick

单位：美国纽约医学院　纽约眼耳鼻喉科医院

关键词：近视；病理生理学；生长因子；病理生理学；视网膜色素上皮细胞；病理生理学；黑素细胞；病理生理学；成纤维细胞；病理生理学

　　［摘　要］　目的:探讨人眼视网膜色素上皮细胞(RPE)、黑素细胞、成纤维细胞与碱性成纤维细胞生长因子(bFGF)、β转化生长因子(TGF-β)和肝细胞生长因子(HGF)的关系。方法:①用不含血清的培养液培养三种细胞48h，收集培养液，用酶标免疫吸附法测量生长因子含量；②将各种生长因子加入含有三种不同细胞的培养液中，五天后行细胞计数。 结果:在RPE和成纤维细胞培养液中能测出HGF、bFGF和TGF，而在黑素细胞培养液中则不能测出。bFGF能刺激上述三种细胞的生长，HGF能刺激RPE与黑素细胞的生长，而TGF-β能抑制三种细胞的生长。结论：RPE、成纤维细胞和黑素细胞具有上述生长因子受体，其中RPE和成纤维细胞还能产生上述生长因子。视网膜产生的各种与近视有关的信使不能透过RPE—脉络膜，所以不能直接作用于巩膜。RPE、成纤维细胞和黑素细胞通过产生、抑制和应答各种生长因子、神经递质及激素，在近视发生中产生作用。

　　［中图分类号］　R778.1⁺1　　　［文献标识码］　A

　　［文章编号］　1008-1801(2000)04-0197-04

Role of RPE-Choroid axis on the occurrence of myopia

HU Dan-ning,Steven A. McCormick.

　　(Tissue Culture Center, New York Eye and Ear Infirmary, New York Medical College)

　　Abstract：　Objective：To study the production of and response to three growth factors related to the occurrence of myopia basic fibroblast growth factor (bFGF), transforming growth factor-β(TGF-β) and hepatocyte growth factor (HGF) by the cell components of RPE-choroid axis (RPE, melanocytes and fibroblasts) in vitro.Methods：RPE, uveal melanocytes and choroidal fibroblasts were isolated and cultured as previously reported. ①Early passages of cells were cultured with serum-free medium for 48 hours. The conditioned media were collected and the amounts of various growth factors were measured by immunoassay. ②Various growth factors were added to the culture media of these cells. After 5 days, cells were counted and compared to the controls.Results：HGF(37-625pg/ml)，bFGF(5-34pg/ml) and TGF-β(359-3271pg/ml) were detected in the conditioned media of RPE and fibroblasts, but not in that of melanocytes. bFGF stimulated the growth of RPE, fibroblasts and melanocytes. HGF stimulated the growth of RPE and melanocytes. TGF-β inhibited the growth of all these cells.Conclusion：RPE, fibroblasts and melanocytes have the receptors of these growth factors. RPE and fibroblasts also can produce these growth factors. The present and previous studies indicate that various retina-originated messengers related to the occurrence of myopia cannot pass through the RPE-choroid without interruption to influence the sclera directly. RPE, fibroblasts and melanocytes play an important role in the occurrence of myopia via the production, binding and response to various growth factors, neurotransmitters and hormones.

　　Key words：　myopia/physiopathology; growth factor/physiopathology; retinal pigment epithelium(RPE)/physiopathology; melanocyte/physiopathology; fibroblast/physiopathology

　　视网膜色素上皮(retinal pigment epithelium,RPE)—脉络膜在近视发病中所起的作用，历来很少有人研究^［1］。实验动物近视研究中发现诱使近视发生的物质主要来自神经视网膜。在近视形成过程中，各种视网膜产生的生化物质(如神经递质、激素、生长因子等)的制造会有所增加或减少^［2～4］。过去一般认为此类生化物质(可称为近视信使)能畅行无阻地通过RPE和脉络膜到达巩膜，从而促进或抑止近视之生成。但我们的研究发现脉络膜黑素细胞(melanocyte)具有多种近视信使之受体，从而可阻断其作用^［5，6］。因此我们(1998)提出一假设，即近视信使从视网膜产生后先作用于黑素细胞，产生新的信使再作用于巩膜，影响近视之发生^［1］。

　　在已知的近视信使中有两种生长因子可影响实验近视之发生，即碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)与β转化生长因子(transforming growth factor-β,TGF-β)^{［2，3，7］}。在分子生物学研究中又发现肝细胞生长因子(hepatocyte growth factor, HGF)能影响眼球之生长^［8］。

　　我们已建立了人眼RPE、脉络膜黑素细胞与成纤维细胞的分离培养方法及体外试验模型^［9～13］。本研究旨在探讨：①体外培养中以上三种细胞制造bFGF、HGF、TGF-β之能力。②该三种生长因子对上述三种细胞之作用。

　　1　对象和方法

　　人眼RPE细胞按我们已报告之方法进行分离培养，用加有10%胎牛血清及加2mM谷氨酰胺(gltamine)的F12培养液培养^［9］。人眼脉络膜黑素细胞用我们报告的酶梯次分离法进行分离，用加有10%胎牛血清、50ng/ml的12-0-tetradecancyl-phorbol-13-acetate(TPA)、0.1mM的isobutylmethylxanthine与10ng/ml cholera toxin的F12培养液培养^［10］。人眼成纤维细胞系在去除贴附的RPE后用酶分离法从脉络膜分离，用含10%胎牛血清的DMEM培养液培养。细胞鉴定系用免疫细胞化学方法，RPE能表达S-100与细胞角蛋白(cytokeratin)，黑素细胞仅表达S-100，成纤维细胞则两者均不表达。

　　测试生长因子的制造时，用不含血清的培养液培养三种细胞48小时，收集培养液，用酶标免疫吸附测定法(ELISA)测量bFGF、TGF-β2与HGF之含量。测试生长因子作用时，将三种细胞接种到24皿板，每皿接种1×10⁴细胞。次日将三种生长因子分别加入。测试浓度为：bFGF,30ng/ml；HGF，100ng/ml；TGF-β2，3ng/ml。以不加生长因子之培养作对照。培养5天后将细胞脱下，计数并与对照组比较，以t-test测定其差别之显著性。

　　2　结果

　　RPE培养液、成纤维细胞培养液、黑素细胞培养中三种生长因子的浓度见表1。

表1　三种培养液中三种生长因子的浓度　(pg/ml)

　　Tab.1　The concentration of three types of

　　growth factors in the three types of culture

　　bFGF能刺激RPE、黑素细胞及成纤维细胞之生长，HGF能刺激RPE与黑素细胞之生长，TGF-β2能抑制该三种细胞之生长(图1)。

图1　各种生长因子对培养的色素上皮细胞、脉络膜黑素细胞

　　及成纤维细胞的影响。与对照组比较，**P＜0.01

　　Fig.1　Effects of various growth factors on the growth of RPE,

　　uveal melanocytes and fibroblasts in vitro.

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