摘要目的 研究AG490阻断Stat3信号转导通路激活对豚鼠巩膜成纤维细胞MMP-2和Integrinβ1表达的影响.方法 实验研究.选用传3代处于对数生长期豚鼠巩膜成纤维细胞.实验共分4个组,分别为A组(空白对照组):不加胰岛素生长因子1(IGF-1)的DMEM培养液、B组(单纯IGF-1组)、C组(IGF-1+PBS组)、D组(IGF-1+25 μm0l/L AG490组).置入37 ℃ CO2细胞培养箱中培养48 h后终止培养.采用RT-PCR和免疫印迹检测4组中Stat3、MMP-2、Integrinβ1mRNA水平和Stat3、p-Stat3(结合位点Tyr-705)、MMP-2、Integrinβ1的蛋白含量变化.用配对t检验和one-wayANOVA方法对测得的数据作处理分析.结果 A、B、C、D组Stat3蛋白表达量分别为0.0637±0.0024、0.6167±0.0276、0.6141±0.0271、0.0674±0.0044,mRNA表达量分别为0.3600±0.0148、0.6315±0.0003、0.6311±0.0001、0.3702±0.0142;p-Stat3蛋白表达量分别为0.0031±0.0000、0.4252±0.0107、0.4274±0.0040、0.0032±0.0000,MMP2蛋白表达量分别为0.0044±0.0002、0.5252±0.0083、0.5251±0.0076、0.0053±0.0003,mRNA表达量分别为0.3067±0.0196、0.5894±0.0122、0.5858±0.0083、0.3107±0.0082.与A、D组比较,B、C组的Stat3、p-Stat3、MMP-2蛋白和基因转录水平明显上调(tpr=-32.324,-26.284,-32.876,-26.345,-68.668,-58.724,-187.481,-58.842,-110.264,-120.256,-121.345,-120.286;tmRNA=-31.554,-31.178,-31.286,-31.198,-12.076,-14.969,-11.896,-14.546;均P＜0.05),但B、C组间比较差异无统计学意义(tp=-32.720,-32.816,-68.668,-187.481,-110.264,-121.345;tmRNA=-0.692,-0.579;均P＞0.05);而Integrinβ1在4个组中无明显改变(Fpr=0.214;FmRNA=0.045,均P＞0.05).结论 AG490阻断Stat3信号通路激活可下调豚鼠巩膜成纤维细胞MMP-2的表达,AG490可以阻断Stat3信号通路激活,是否可作为预防近视的靶点候选药物有待进一步研究.

abstractsObjective To explore the effect of Blocking activation of IGF-1-Stat3 signaling pathway in guinea pig sclera fibroblast (GSFs) by AG490 on expressions of MMP-2, Integrinβ1. Methods Cultured GSFs were divided into four groups: group A( control group: only DMEM without IGF-1 ), group B (only IGF-1 group), group C( IGF-1 + PBS group), group D( IGF-1 +25 μmol/L AG490 group). The expressions of Stat3, p-Stat3, MMP-2, Integrinβ1 protein induced by IGF-1 and inhibited by AG490 in GSFs were detected by Western blot. The levels of Stat3, MMP-2 and Integrinβ1mRNA were detected by RTPCR. Results Compared with Groups A and D, Stat3, p-Stat3, MMP-2 protein expression in groups B and C were expressed at higher level ( tpr = - 32. 324, - 26. 284, - 32. 876, - 26. 345, - 68. 668, - 58. 724,- 187.481, - 58. 842, - 110. 264, - 120. 256, - 121. 345, - 120. 286; tmRNA = - 31. 554, - 31. 178,- 31. 286, - 31.198, - 12. 076, - 14. 969, - 11. 896, - 14. 546, P ＜ 0. 05 ), but the expression levels were not obviously different between groups B and C (tp = -32.720, -32. 816, -68.668, -187.481,- 110. 264, - 121. 345; tmRNA = - 0. 692, - 0. 579, P ＞ 0. 05 ), which were similar to mRNA level. The Integrinβ1 protein and mRNA were expressed in groups A, B, C and D but no significant difference among them respectively (Fpr =0.214;FmRNA = 0.045,P ＞0.05). Conclusions Activation of Stat3 signaling pathway may be involved in up-modulating the expression of MMP-2 in GSFs, and not affect the Integrinβ1protein and mRNA changes. The results reveal that Stat3 signaling transduction pathway may play a critical role in sclera remodeling by means of modulating MMP-2 expression.