Molecular biological and biochemical study of intraocular pressure-sensing mechanism

我们证明，培养的牛小梁细胞响应 72 小时的循环机械拉伸，产生金属蛋白酶-1 (TIMP-1) 和基质金属蛋白酶-2 (MMP-2) 的组织抑制剂。以 4500 微应变的大小和 30 秒的周期对 6 厘米培养皿中的培养细胞进行循环机械拉伸。人类小梁细胞还表达了一种新基因，以响应 24 小时的循环机械拉伸。该新基因是通过基于抑制聚合酶链式反应的消减杂交分离出来的。从拉伸的小梁细胞和对照小梁细胞中分离信使RNA，并合成cDNA。从拉伸的细胞衍生的 cDNA 中减去对照细胞衍生的 cDNA。将扣除的 cDNA 片段克隆到质粒载体中。通过Northern印迹杂交检查拉伸细胞和对照细胞中每个克隆的表达。然后通过人眼cDNA LIBRARY筛选分离出相应的全长cDNA。这些事实表明小梁细胞对机械刺激有反应，并且小梁细胞中存在眼内传感机制。

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.