Tamm E R, Russell P, Epstein D L, Johnson D H, Piatigorsky J
Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland, USA.
Invest Ophthalmol Vis Sci. 1999 Oct;40(11):2577-82.
To study factors that modulate myocilin/trabecular meshwork inducible glucocorticoid response protein (TIGR) mRNA expression in human trabecular meshwork (TM).
mRNA from fresh TM of four human donors, from perfused anterior segment organ cultured TM of three donors, and from four primary TM cell lines of different donors was isolated. The full length cDNA of myocilin/TIGR was cloned from TM mRNA using a polymerase chain reaction approach and used as probe for northern blot analysis hybridization. Trabecular meshwork cell cultures were treated with transforming growth factor (TGF)-beta1 (1 ng/ml), dexamethasone (10(-7) M), and mechanical stretch (10%).
mRNA for myocilin/TIGR could be readily detected by northern blot analysis hybridization in 2 to 3 microg of total RNA from all fresh and all organ-cultured TM samples. In contrast, no mRNA for myocilin/TIGR could be detected in 20 microg of total RNA isolated from three different primary TM cell lines. Only one TM cell line had a baseline expression of myocilin/TIGR, which was 35- to 55-fold lower than that of fresh or organ-cultured TM samples. Treatment of TM cell cultures with dexamethasone for 1 day markedly increased expression of myocilin/TIGR mRNA, an effect that was even more pronounced after 3 days of treatment. Treatment with TGF-beta1 for 24 hours had no effect; however, after 3 and 12 days of treatment a 3.8- and 4-fold increase in myocilin/TIGR mRNA expression was observed. Expression of myocilin/TIGR mRNA was also increased after 10% mechanical stretch; however, in contrast to the effects of TGF-beta-1, this effect was observed much earlier (8-24 hours) after treatment.
Dynamic mechanical stimuli maintain myocilin/TIGR expression in TM in situ and lack of these stimuli in monolayer cell cultures might be involved in downregulation of myocilin/TIGR expression.
研究调节人小梁网（TM）中肌纤蛋白/小梁网诱导糖皮质激素反应蛋白（TIGR）mRNA表达的因素。
从四名人类供体的新鲜TM、三名供体的灌注眼前节器官培养TM以及四名不同供体的原代TM细胞系中分离mRNA。使用聚合酶链反应方法从TM mRNA中克隆肌纤蛋白/TIGR的全长cDNA，并用作Northern印迹分析杂交的探针。小梁网细胞培养物用转化生长因子（TGF）-β1（1 ng/ml）、地塞米松（10⁻⁷ M）和机械拉伸（10%）处理。
通过Northern印迹分析杂交在所有新鲜和所有器官培养的TM样品的2至3 μg总RNA中可轻易检测到肌纤蛋白/TIGR的mRNA。相比之下，从三种不同的原代TM细胞系分离的20 μg总RNA中未检测到肌纤蛋白/TIGR的mRNA。只有一个TM细胞系有肌纤蛋白/TIGR的基线表达，比新鲜或器官培养的TM样品低35至55倍。用地塞米松处理TM细胞培养物1天可显著增加肌纤蛋白/TIGR mRNA的表达，处理3天后这种效果更明显。用TGF-β1处理24小时没有效果；然而，处理3天和12天后，观察到肌纤蛋白/TIGR mRNA表达分别增加3.8倍和4倍。10%机械拉伸后肌纤蛋白/TIGR mRNA的表达也增加；然而，与TGF-β1的作用相反，这种作用在处理后更早（8至24小时）观察到。
动态机械刺激维持TM原位的肌纤蛋白/TIGR表达，单层细胞培养中缺乏这些刺激可能参与肌纤蛋白/TIGR表达的下调。
