虹膜色素颗粒培养的牛小梁细胞中转化生长因子β1和纤维连接蛋白表达的变化及其意义

Expression changes of transforming growth factor-β1 and fibronectin in bovine trabecular meshwork cells cultured by iris pigment particles

背景色素性青光眼和假性剥脱性青光眼均以色素颗粒沉积于房角为主要特征，虹膜色素颗粒的沉积除阻塞房角的房水引流通道外，对小梁网的吞噬功能也可造成不可逆性损害，从而导致细胞外基质重塑异常。青光眼患者房水和小梁网组织中转化生长因子-β（TGF-β）含量升高，尤以假性剥脱性青光眼患者更为明显，但TGF-β和纤维连接蛋白（FN）在虹膜色素颗粒培养的小梁细胞中的表达变化尚不清楚。目的探讨虹膜色素颗粒培养的牛小梁细胞中TGF-β1和FN的表达变化，为青光眼的发病机制研究提供实验依据。方法采用组织块培养法培养新鲜牛眼小梁细胞，根据细胞的形态对培养细胞进行鉴定。将第3代牛小梁细胞接种于6孔培养板并分为正常对照组和虹膜色素颗粒干预组，分别在培养基中加入100μl色素颗粒工作液（终浓度为1×10^7／m1）和100μl 0．01mol／L PBS，继续培养24h。采用荧光定量PCR法检测各组细胞内TGF-β1 mRNA和FN mRNA的相对表达量；采用竞争ELISA法检测各组细胞培养上清液中TGF-β1和FN蛋白的质量浓度。结果体外培养的细胞呈长梭形、多角形或树枝状，细胞中可见色素，细胞核圆且居中，符合牛小梁细胞的形态特征。荧光定量PCR法检测显示，虹膜色素颗粒干预组细胞中TGF-β1 mRNA相对表达量为2．98±0.27，明显高于正常对照组的1．00±0.00，FN mRNA相对表达量为0．36±0.10，明显低于正常对照组的1．000±0．000，差异均有统计学意义（t=12．68，P=0．00；t=10．60，P=0．00）。ELISA法检测显示，虹膜色素颗粒干预组细胞培养上清液中TGF-β1蛋白质量浓度为（156．60±9．74）ng／L，明显高于正常对照组的（65．46±14．24）ng／L，FN蛋白质量浓度为（59．29±15．79）mg／ml，低于正常对照组的（102．10±12．14）mg／ml，差异均有统计学意义（t=9．15，P=0．00；t=3．72，P=0．02）。结论虹膜色素颗粒培养的牛小梁细胞中TGF-β1表达上调而FN表达下调，推测TGF-β1和FN参与色素性和假性剥脱性青光眼的发病和进展过程。

Background Pigmentary glaucoma and pseudoexfoliation glaucoma arc characterized by pigment dispersion in trabecular meshwork, and the dipersitional pigment probably contributes to the resistance of aqueous outflow pathway, irreversible damage of the trabecular meshwork and the remodeling abnormality of extracellular matrix. Researches determined that contents of transforming growth factor-β（TGF-β） in the aqueous humor are increased in glaucomatous eyes. However,the effects of TGF-β and fibroneetin （FN） in the trabecular meshwork cells （TMCs） acted by iris pigmentary particles still are not elucidated. Objective This study was to investigate the effects of iris pigment particles on TGF-β1 and FN expression in bovine TMCs （BTMCs） cultured in vitro. Methods BTMCs were cultured in vitro by explant culture method and identified by morphological evaluation. The third generation of BTMCs were divided into normal control group and pigment group, and 100 μl PBS and 100μl iris pigment suspension （final concentration of 1 × 10^7 partieles/ml） were added into the medium for 24 hours,respectively. The expressions of TGF-β1 mRNA, FN mRNA and their proteins in the BTMCs were assayed by real-time fluorescence quantitative PCR and ELISA,respectively. Results Cultured cells grew well and showed the fusiform, polygon and dendritic like in shape,with pigmented and round nuclei. The relative expression levels of TGF-β1 mRNA and FN mRNA in the cells were 2.98±0.27 and 0.36±0.10 in the iris pigment group,which were significantly higher than 1. 00±0. 00 and 1.00 ±0. 00 in the normal control group （t = 12. 68,10. 60, both at P = 0.00）. The concentrations of TGF-β1 protein and FN protein in the cell suspension were （156.60±9.74）ng/L and （59.29±15.79）mg/ml in the iris pigment group, showing significant differences in comparison with （65.46±14. 24） ng/L and （ 102. 10 ± 12.14）mg/ml in the normal control group （t=9.15,P=0.00;t=3.72,P=0. 02）. Conclusions The expression of TGF-β1 is up-regulated and that of FN is down-regulated in BTMCs cultured by iris pigment,inferring that TGF-β1 and FN participate in the pathogenesis and development of pigmentary and pseudoexfoliation glaucoma.