Y-27632对眼Tenon囊成纤维细胞中转化生长因子β1信号转导的干扰作用
Interference of Y-27632 on the signal transduction of transforming growth factor beta type 1 in ocular Tenon capsule fibroblasts.
作者信息
Zhang Xiao-Hui, Sun Nai-Xue, Feng Zhao-Hui, Wang Chao, Zhang Yi, Wang Jian-Ming
机构信息
Department of Ophthalmology, the Second Affiliated Hospital, College of Medicine, Xi'an Jiaotong University, Xi'an 710004, Shaanxi Province, China.
出版信息
Int J Ophthalmol. 2012;5(5):576-81. doi: 10.3980/j.issn.2222-3959.2012.05.06. Epub 2012 Oct 18.
AIM
To investigate the interfering effect of Y-27632, a ROCK-I selective inhibitor, on the signal transduction pathway of transforming growth factor-β1 (TGF-β1) in ocular Tenon capsule fibroblasts (OTFS) in vitro.
METHODS
After OTFS from passages 4 to 6 in vitro were induced by TGF-β1 and then treated by Y-27632, the changes of the OTFS cell cycles were analyzed via flow cytometry, and the proteins expression of the α-smooth muscular actin (α-SMA), connective tissue growth factor (CTGF), collagen I were calculated by Western blot. After OTFS treated by the different concentrations of Y-27632, the expression levels of the α-SMA, CTGF and collagen I mRNA were assayed by RT-PCR.
RESULTS
Y-27632 had no markedly effect on the OTFS cell cycles. After treated by TGF-β1, OTFS in G1 period significantly increased. The cell cycles distribution by both TGF-β1 and Y-27632 had no remarkable difference from that in control group. Y-27632 significantly inhibited the proteins expressions of both α-SMA and CTGF, while to some extent inhibited that of collagen I. TGF-β1 significantly promoted the proteins expressions of α-SMA, CTGF and collagen I. After OTFS treated by both TGF-β1 and Y-27632, of α-SMA, the protein expression was similar with that in control group (P=0.066>0.05), but the protein expression of CTGF or collagen I, respectively, was significantly different from that in control group (P=0.000<0.01). The differences of expressions of the α-SMA, CTGF and collagen I mRNA in 30, 150, 750µmol/L Y-27632 group were statistically significant, compared with those in control group, respectively (α-SMA, P=0.002, 0.000, 0.000; CTGF, P=0.014, 0.002, 0.001; collagen I, P=0.003, 0.002, 0.000).
CONCLUSION
Blocking the Rho/ROCK signaling pathway by using of Y-27632 could inhibit the cellular proliferation and the expression of both CTGF and α-SMA whatever OTFS induced by TGF-β1 or not. Y-27632 suppressed the expression of collagen I mRNA without induction.
目的
研究ROCK-I选择性抑制剂Y-27632对体外培养的眼Tenon囊成纤维细胞（OTFS）中转化生长因子-β1（TGF-β1）信号转导通路的干扰作用。
方法
体外培养的第4至6代OTFS经TGF-β1诱导后再用Y-27632处理，通过流式细胞术分析OTFS细胞周期的变化，采用蛋白质免疫印迹法检测α-平滑肌肌动蛋白（α-SMA）、结缔组织生长因子（CTGF）、Ⅰ型胶原的蛋白表达。不同浓度Y-27632处理OTFS后，采用逆转录-聚合酶链反应（RT-PCR）检测α-SMA、CTGF和Ⅰ型胶原mRNA的表达水平。
结果
Y-27632对OTFS细胞周期无明显影响。TGF-β1处理后，G1期OTFS显著增加。TGF-β1与Y-27632共同处理后的细胞周期分布与对照组无显著差异。Y-27632显著抑制α-SMA和CTGF的蛋白表达，对Ⅰ型胶原的表达有一定程度的抑制作用。TGF-β1显著促进α-SMA、CTGF和Ⅰ型胶原的蛋白表达。TGF-β1与Y-27632共同处理后的OTFS，α-SMA的蛋白表达与对照组相似（P=0.066>0.05），但CTGF和Ⅰ型胶原的蛋白表达与对照组相比有显著差异（P=0.000<0.01）。30、150、750μmol/L Y-27632组α-SMA、CTGF和Ⅰ型胶原mRNA表达与对照组相比差异有统计学意义（α-SMA，P=0.002、0.000、0.000；CTGF，P=0.014、0.002（此处原文可能有误，按照逻辑应为0.002）、0.001；Ⅰ型胶原，P=0.003、0.002、0.000）。
结论
使用Y-27632阻断Rho/ROCK信号通路，无论OTFS是否由TGF-β1诱导，均可抑制细胞增殖以及CTGF和α-SMA的表达。Y-27632在无诱导情况下抑制Ⅰ型胶原mRNA的表达。
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