Molecular biological and biochemical study of intraocular pressure-sensing mechanism

我们证明，培养的牛眼小梁细胞产生的金属蛋白酶组织抑制剂-1（TIMP-1）和基质金属蛋白酶-2（MMP-2）的周期性机械拉伸72小时。以4500微应变的量级和30秒的周期对6-cm培养皿中的培养细胞施加循环机械拉伸。人类小梁细胞也表达了一种新的基因，以响应24小时的周期性机械拉伸。该基因是通过抑制性聚合酶链反应的消减杂交技术分离得到的。从伸展的小梁细胞和对照小梁细胞中分离信使RNA，并合成cDNA。从拉伸的细胞来源的cDNA中减去对照细胞来源的cDNA。将消减的cDNA片段克隆到质粒载体中。通过北方印迹杂交检测拉伸细胞和对照细胞中每个克隆的表达。然后通过人眼cDNA文库的筛选分离相应的全长cDNA。这些事实表明，小梁细胞对机械刺激作出反应，并且小梁细胞中存在眼内感知机制。

We demonstrated that cultured bovine trabecular cells produced tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinase-2 (MMP-2) in response to cyclic mechanical stretching for 72 hours. Cyclic mechanical stretching was applied to cultured cells in 6-cm petri dishes at the magnitude of 4500 microstrain and at a cycle of 30 seconds. Human trabecular cells also expressed a novel gene in response to cyclic mechanical stretching for 24 hours. This new gene was isolated by subtractive hybridization based on suppression polymerase chain reaction. Messenger RNA was isolated from stretched trabecular cells and control trabecular cells, and cDNA was synthesized. Control cell-derived cDNA was subtracted from stretched cell-derived cDNA. The subtracted cDNA fragments were cloned into a plasmid vector. The expression of each clone in stretched cells and in control cells was examined by Northern blot hybridization. The corresponding full-length cDNA was then isolated by screening of human eye cDNA LIBRARY. These facts suggest that trabeuclar cells respond to mechanical stimuli and that an intraocular-sensing mechanism is present in trabecular cells.