Molecular biological and biochemical study of intraocular pressure-sensing mechanism

我们证明了培养的牛眼小梁细胞在周期性机械拉伸72小时后产生了金属蛋白酶组织抑制因子-1(TIMP-1)和基质金属蛋白酶-2(MMP-2)。在6 cm培养皿中对培养细胞进行循环机械拉伸，应变大小为4500微应变，周期为30秒。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。该新基因是通过抑制性聚合酶链式反应的消减杂交技术分离出来的。从拉伸的小梁细胞和对照的小梁细胞中提取信使RNA，合成cDNA。从拉伸的细胞来源的基因中减去对照细胞来源的基因。将消减后的cDNA片段克隆到载体上。Northern印迹杂交检测各克隆在拉伸细胞和对照细胞中的表达。通过对人眼cDNA文库的筛选，获得相应的全长c DNA。这些事实表明，小梁细胞对机械刺激有反应，在小梁细胞中存在眼内感觉机制。

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.