Tripathi Brenda J, Tripathi Ramesh C, Chen Jianguang, Gotsis Stelios, Li Junping
Department of Pathology and Microbiology, University of South Carolina School of Medicine, 6439 Garners Ferry Road, Columbia, SC 29209, USA.
Exp Eye Res. 2004 Mar;78(3):653-60. doi: 10.1016/j.exer.2003.09.011.
We investigated the mRNA and protein expression of fibronectin and stromelysin-1 (matrix metalloproteinase-3, MMP-3) by trabecular cells treated with growth factors present in primary and secondary aqueous humors. Serum-deprived trabecular cells were incubated for 48 hr or 7 days in medium containing either primary or secondary aqueous humor growth factors or in serum-free medium. We extracted total RNA, performed reverse transcription-polymerase chain reaction using primer pairs for fibronectin, stromelysin-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and quantified the products. We utilized Western blotting to detect and quantify fibronectin and stromelysin-1 protein. Compared to controls, expression of fibronectin mRNA by trabecular cells was increased by 50 and 100% after incubation in primary aqueous humor growth factors for 48 hr or 7 days, respectively, and 50 and 130% after incubation in secondary aqueous humor growth factors. Stromelysin-1 mRNA expression was decreased by 25 and 50% after incubation in primary aqueous humor growth factors for 48 hr or 7 days, respectively, and 80 and 85% after incubation for 48 hr or 7 days, respectively, in secondary aqueous humor growth factors. Fibronectin protein increased 3.5-fold and 6-fold after incubation for 48 hr with primary or secondary aqueous humor growth factors, respectively; after 7 days, the level increased 4- and 7-folds, respectively. Stromelysin-1 protein was not detectable by western blotting. The up-regulation of fibronectin mRNA by trabecular cells exposed to growth factors present in secondary aqueous humor augmented by the down-regulation of stromelysin-1 mRNA contributed to the accumulation of fibronectin. Our findings open the possibility that induction of stromelysin-1 gene expression in the trabecular meshwork of glaucomatous eyes could effectively reduce buildup of fibronectin in the aqueous outflow pathway to decrease outflow resistance in glaucomatous states of the eye.
我们研究了小梁细胞在原发性和继发性房水中生长因子作用下纤连蛋白和基质溶解素-1（基质金属蛋白酶-3，MMP-3）的mRNA和蛋白质表达。将血清饥饿的小梁细胞在含有原发性或继发性房水生长因子的培养基或无血清培养基中孵育48小时或7天。我们提取总RNA，使用针对纤连蛋白、基质溶解素-1和甘油醛-3-磷酸脱氢酶（GAPDH）的引物对进行逆转录聚合酶链反应并对产物进行定量。我们利用蛋白质免疫印迹法检测和定量纤连蛋白和基质溶解素-1蛋白。与对照组相比，小梁细胞在原发性房水生长因子中孵育48小时或7天后，纤连蛋白mRNA表达分别增加50%和100%，在继发性房水生长因子中孵育后分别增加50%和130%。在原发性房水生长因子中孵育48小时或7天后，基质溶解素-1 mRNA表达分别降低25%和50%，在继发性房水生长因子中孵育48小时或7天后分别降低80%和85%。在原发性或继发性房水生长因子中孵育48小时后，纤连蛋白蛋白分别增加3.5倍和6倍；7天后，水平分别增加4倍和7倍。通过蛋白质免疫印迹法未检测到基质溶解素-1蛋白。暴露于继发性房水中生长因子的小梁细胞中纤连蛋白mRNA的上调以及基质溶解素-1 mRNA的下调导致了纤连蛋白的积累。我们的研究结果揭示了一种可能性，即诱导青光眼眼中小梁网中基质溶解素-1基因的表达可能有效减少房水流出途径中纤连蛋白的积聚，从而降低青光眼状态下的眼内流出阻力。
