陈小玲, 何为民, 李 薇.IGF-1R对甲状腺相关眼病眼眶成纤维细胞合成HA的影响及其作用机制.四川大学学报(医学版),2017,48(5):727-731
IGF-1R对甲状腺相关眼病眼眶成纤维细胞合成HA的影响及其作用机制
Effects and Its Mechanism of IGF-1R on the Synthesis of Hyaluronic Acid in Orbital Fibroblasts of
  
中文关键词:  甲状腺相关眼病 透明质酸 胰岛素样生长因子受体 信号通路
英文关键词:Thyroid associated ophthalmopathy Hyaluronic acid Insulin-like growth factor-1 receptor Signal pathway
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中文摘要:
      目的 探讨胰岛素样生长因子1受体(IGF1R)对甲状腺相关眼病(TAO)眼眶成纤维细胞合成透明质酸(HA)的影响及其作用的信号通路。方法 体外培养19例(23只眼)TAO患者(TAO组)及5例(5只眼)正常对照(对照组)眼眶成纤维细胞,并采用免疫荧光染色进行鉴定;细胞培养液中分别加入不同浓度重组人IGF1,ELISA检测细胞培养上清液中HA质量浓度;细胞培养液中分别加入IGF1受体阻断剂1H7、磷脂酰肌醇3激酶(PI3K)阻断剂LY294002或蛋白激酶B(PKB)抑制剂(即Akt抑制剂)Ⅳ对细胞进行预处理,再加入重组人IGF1,ELISA检测培养上清液中HA质量浓度,Western blot检测PI3K、Akt及pAkt蛋白表达情况。结果 TAO组HA质量浓度在重组人IGF1 0~5 nmol/L处理时逐渐增加,在IGF1 10 nmol/L时达到最大值,随后在IGF1 20~50 nmol/L时出现波动。与正常对照组相比,0~50 nmol/L重组人IGF1处理的TAO组HA质量浓度均增高,差异有统计学意义( P<0.01)。IGF1受体阻断剂1H7和Akt抑制剂Ⅳ对细胞预处理后,再加入重组人IGF1,其细胞培养上清液中的HA质量浓度较对照组降低,差异有统计学意义( P<0.01),PI3K阻断剂LY294002预处理后,细胞培养上清液中HA质量浓度约有降低,但差异无统计学意义( P=0.390)。与空白对照组相比,IGF1处理组细胞pAkt蛋白表达水平升高,而对PI3K和总Akt蛋白表达无明显影响;1H7和LY294002可降低PI3K及pAkt蛋白的表达水平,对总Akt蛋白表达的抑制作用不明显;Akt抑制剂Ⅳ处理组,PI3K、Akt及pAkt蛋白表达水平均较对照组明显降低。结论 TAO患者眼眶成纤维细胞合成HA增加,IGF1能促进HA合成,且这种促进作用部分通过PI3K/Akt信号通路实现。
英文摘要:
      【Abstract】 Objective To explore the effects of insulin-like growth factor1 (IGF1) receptor (IGF1R) on the synthesis of hyaluronic acid (HA) in orbital fibroblasts of thyroid associated ophthalmopathy (TAO) and its signal pathway. Methods Orbital fibroblasts were harvested from TAO ( n=19) and normal control ( n=5), then were primary cultured and treated with recombinant human IGF1 in different concentrations. Before the treatment of IGF1, the cells were pretreated respectively with 1H7, LY294002 or AKT inhibitor Ⅳ for 24 h. The production of HA was measured using a commercial ELISA kit, and the synthesis of PI3K, Akt and pAkt was measured by Western blot. Results Along with the increase of recombinant human IGF1 concentration, the synthesis of HA by TAO orbital fibroblasts were increased as well,the synthesis of HA peaked at the concentration of 10 nmol/L in IGF1 in TAO group ( P<0.01). Compared with the normal control, orbital fibroblasts from TAO had the synthesis of HA increased after the treatment of IGF1 ( P<0.01). The pretreatment of 1H7 or AKT inhibitor Ⅳ significantly decreased the HA concentration in culture media ( P<0.01), while the decrease of HA synthesis in the group pretreated with LY294002 was not statistically significant ( P=0.390). IGF1 treatment increased the level of pAkt expression, but it seems no effects on PI3K and Akt expression. 1H7 and LY294002 decreased the expression of PI3K and pAkt protein, but no obvious inhibitory effect on total Akt protein. Akt inhibitor Ⅳ decreased the expression of PI3K, Akt and pAkt. Conclusion The synthesis of HA by orbital fibroblasts could be increased in TAO, which may partially via phosphoinositide 3kinase/Akt pathway.
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