Protein expression in human trabecular meshwork: downregulation of RhoGDI by dexamethasone in vitro
Protein expression in human trabecular meshwork: downregulation of RhoGDI by dexamethasone in vitro
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发表时间：
2010-02
期刊：
影响因子：
2.2
通讯作者：
Minbin Yu;Jingjing Sun;W. Peng;Ziyan Chen;Xianchai Lin;Xuyang Liu;Ming-tao Li;Kaili Wu
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作者：
Minbin Yu;Jingjing Sun;W. Peng;Ziyan Chen;Xianchai Lin;Xuyang Liu;Ming-tao Li;Kaili Wu
人类小梁网（TM）蛋白质组的表征是了解其在正常和青光眼条件下的作用的有价值的一步。本研究利用蛋白质组学技术研究正常人TM中表达的一组蛋白，并鉴定在体外地塞米松（DEX）处理TM细胞（TMCs）后表达的差异蛋白。方法从人供眼组织中分离出TM组织，并进行汇总。用或不加DEX培养永生化的人tmc。通过双向电泳（2-DE）分离蛋白质提取物。将TMT凝胶中的蛋白斑点切除、染色，进行凝胶内胰蛋白酶消化，并用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF-MS）进行鉴定。为了确定糖皮质激素对TMCs表达模式的影响，我们用DEX处理TMCs，并用3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四唑（MTT）染料和2-DE进行检测。通过western blotting和免疫细胞学染色验证了差异表达蛋白RhoGDI。结果在2-DE凝胶上显示，综合蛋白集包括来自TMT和TMCs的850多个蛋白点。在TMT凝胶中成功鉴定了235个点。鉴定的蛋白功能类别主要包括代谢过程、细胞粘附、抗凋亡、细胞运动、碳水化合物代谢过程、信号转导和转录调节。MTT实验证明，在DEX治疗的3天内，TMCs的增殖受到时间和剂量依赖性的抑制。在培养48 h的细胞组中，通过2-DE、western blotting和免疫细胞学染色检测，RhoGDI表达降低。相反，RhoA （RhoGDI的靶点）的表达在DEX治疗后增加。结论采用经典的蛋白质组学工作流程，对正常人TMT的主要蛋白质补体进行了检测、鉴定和分类。证实了DEX对tmc中RhoGDI表达的抑制作用。
Purpose The characterization of the human trabecular meshwork (TM) proteome is a valuable step toward understanding its role under normal and glaucomatous conditions. This study uses proteomic techniques to investigate the set of proteins expressed in normal human TM and to identify those differentially expressed in response to dexamethasone (DEX) treatment of TM cells (TMCs) in vitro. Methods TM tissue (TMT) was isolated from human donor eyes and pooled. Immortalized human TMCs were cultured with or without DEX. Protein extracts from each were separated by two-dimensional electrophoresis (2-DE). Protein spots in TMT gel were excised, destained, and subjected to in-gel tryptic digestion and identification with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). To determine those proteins whose expression patterns were affected by glucocorticoids, TMCs were treated with DEX and assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) dye and 2-DE. A differentially expressed protein, RhoGDI, was validated by both western blotting and immunocytological staining. Results The comprehensive protein set included more than 850 protein spots from both the TMT and TMCs, as visualized on 2-DE gel. Two-hundred-and-thirty-five spots were successfully identified in the TMT gel. The functional categories of the identified proteins were mainly comprised of metabolic process, cell adhesion, anti-apoptosis, cell motility, carbohydrate metabolic process, signal transduction, and regulation of transcription. During three days of DEX treatment, TMCs’ proliferation was inhibited in a time- and dose-dependent manner, as evidenced by MTT assay. In the 48 h cultured cell group, RhoGDI expression was reduced, as detected by 2-DE, western blotting, and immunocytological staining. In contrast, the expression of RhoA, a target of RhoGDI, increased in response to DEX treatment. Conclusions Using the classic proteomic workflow, the main protein complement of normal human TMT was detected, identified, and categorized. The DEX inhibition of RhoGDI expression in TMCs was evidenced.