SB431542和TGF-β_1对甲状腺相关性眼病患者眼眶成纤维细胞α-SMA及CTGF基因表达的影响  

Effects of SB431542 and TGF-β_1 on α-SMA and CTGF gene expression in orbital fibroblast of patients with thyroid-associated ophthalmolopathy

作　　者：陆燕[1] 黄媛媛[2] 蔡季平[3] 程金伟[3] 魏锐利[3] 

机构地区：[1]南京军区南京总医院眼科,江苏省南京市210002 [2]江苏省口腔医院口腔颌面外科,江苏省南京市210002 [3]第二军医大学附属长征医院眼科,上海市200003

出　　处：《眼科新进展》2015年第3期210-213,共4页Recent Advances in Ophthalmology

基　　金：国家自然科学基金(编号:81200719;81070758);中国博士后科学基金面上资助项目(编号:2013M542579);中国博士后科学基金特别资助项目(编号:2014T71013)~~

摘　　要：目的探讨转化生长因子-β1（transforming growth factor-β1,TGF-β1）和SB431542（TGF-β1受体酶抑制剂）对甲状腺相关性眼病（thyroid-associated ophthalmopathy,TAO）患者眼眶成纤维细胞α平滑肌激动蛋白（α-smooth muscle actin,α-SMA）及结缔组织生长因子（connective tissue growth factor,CTGF）基因表达的影响,为TAO眼外肌纤维化的治疗寻找潜在的药物治疗靶点。方法实验分为3组。第1组：采用实时荧光定量逆转录聚合酶链反应（real-time PCR,RT-PCR）检测不同浓度TGF-β1（0μg·L-1、1μg·L-1、5μg·L-1、10μg·L-1、20μg·L-1）刺激TAO患者眼眶成纤维细胞后α-SMA及CTGF mRNA的表达情况;第2组：采用RT-PCR检测10μg·L-1 TGF-β1在不同的时间点（0 h、3 h、6 h、12 h、24 h、48 h）刺激TAO患者眼眶成纤维细胞后α-SMA及CTGF mRNA的表达情况;第3组（分为4小组处理）：眼眶成纤维细胞不加任何干预、使用30μmol·L-1SB431542刺激眼眶成纤维细胞、10μg·L-1 TGF-β1单独刺激眼眶成纤维细胞、30μmol·L-1 SB431542联合10μg·L-1 TGF-β1刺激眼眶成纤维细胞（SB431542预先刺激眼眶成纤维细胞1 h,然后再加入TGF-β1）,采用RT-PCR检测α-SMA及CTGF mRNA的表达。结果TGF-β1在一定的浓度范围内（1~10μg·L-1）以剂量依赖的方式诱导眼眶成纤维细胞表达α-SMA mRNA及CTGF mRNA,随着浓度增加mRNA表达增加,各浓度组（1μg·L-1、5μg·L-1、10μg·L-1、20μg·L-1）与空白对照组（0μg·L-1）相比,差异均有统计学意义（均为P〈0.05）。TGF-β1在一定的时间范围内（0~24 h）以时间依赖的方式诱导眼眶成纤维细胞表达α-SMA mRNA及CTGF mRNA,随着时间延长mRNA表达增加,各时间组（3 h、6 h、12 h、24 h、48 h）与0 h相比,差异均有统计学意义（均为P〈0.05）。10μg·L-1的TGF-β1诱导眼眶成纤维细胞表达α-SMA mRNA在24 h达到峰值,而CTGF mRNA在12 h即可达到峰值。SB431542对眼眶成纤维细胞α-SMA mRNA及CTGObjective To explore the inhibitive effects of SB431542 and transforming growth factor-β1（ TGF-β1） on α-smooth muscle actin（ α-SMA） and connective tissue growth factor（ CTGF） gene expression in orbital fibroblast of patients with thyroid-associated ophthalmolopathy（ TAO）, and identify the potential pharmacologic target. Methods The samples were divided into three groups. In the first group, the orbital fibroblast were treated with different concentrations（ 0 μg·L-1,1μg·L-1,5μg·L-1, 10 μg·L-1, 20 μg·L-1） of TGF-β1 for 24 hours, and realtime quantitative RT-PCR was performed to observe the effects of TGF-β1 on the expression ofα-SMA and CTGF mRNA. In the second group, the orbital fibroblast were treated with 10 μg·L-1 TGF-β1 at different time points（ 0 hour,3 hours,6 hours, 12 hours, 24 hours, 48 hours）, and real-time quantitative RT-PCR was performed to observe the effects of TGF-β1 on the expression of α-SMA and CTGF mRNA. In the third group, the orbital fibroblast were treated with SB431542（ 30 μmol·L-1） only or 10 μg·L-1 TGF-β1 only or containing 10 μg·L-1 TGF-β1 and SB431542（ 30 μmol·L-1） was added. The cells were pretreated with SB431542 for 1 hour before induction with 10 μg·L-1 TGF-β1, and real-time quantitative RT-PCR was performed to observe the effects of TGF-β1 on the expression of α-SMA and CTGF mRNA. Results The orbital fibroblast induced by TGF-β1 in vivo showed an increasing expression of α-SMA and CTGF mRNA in a dose dependent manner from 1 μg·L-1 to 10 μg·L-1. Other groups（ 1 μg·L-1,5μg·L-1, 10 μg·L-1 and 20 μg·L-1） compared with blank control group（ 0 μg·L-1）, the differences were significant（ all P〈0. 05）. The orbital fibroblast induced by TGF-β1 in vivo showed an increasing expression of α-SMA and CTGF mRNA in a time dependent manner from 0hour to 24 hours. Other groups（ 3 hours,6 hours,12 hours,24 hours and 48 hours） compared with blank control group（ 0 hour）, the differences were signi

关 键 词：甲状腺相关性眼病 转化生长因子-β1 眼眶成纤维细胞转分化 结缔组织生长因子 SB431542 

分 类 号：R771.3[医药卫生—眼科]