多西环素对TGF-β1诱导的人角膜基质细胞纤维化的抑制作用

Inhibitory effect of doxycycline on TGF-β1-induced human corneal stromal cell fibrosis

目的:探讨多西环素(Doxy)对转化生长因子-β1(TGF-β1)诱导的人角膜基质细胞纤维化的抑制作用。方法:收集飞秒激光微小切口基质透镜取出术(SMILE术)后角膜基质透镜提取人角膜基质细胞,利用TGF-β1诱导人角膜基质细胞建立体外角膜纤维化模型,设置对照组、TGF-β1组、DOXY组。采用CCK-8法检测各组细胞增殖能力、划痕实验检测增殖能力;酶联免疫吸附实验(ELISA)测定胶原合成关键因子羟脯氨酸(HYP)含量;实时荧光定量PCR(RT-qPCR)检测纤维化关键因子Thy-1、Vimentin mRNA表达情况;蛋白免疫印迹(western blotting)检测Thy-1、Vimentin蛋白表达水平。结果:与对照组相比,TGF-β1组能促进人角膜基质细胞细胞增殖、迁移,增加HYP含量、Thy-1和Vimentin的表达(均P<0.05)。与TGF-β1组比较,DOXY组细胞增殖、细胞迁移和胶原合成能力明显降低,Thy-1和Vimentin的表达减少(均P<0.05)。结论:Doxy可抑制TGF-β1诱导的人角膜基质细胞细胞增殖、迁移和胶原合成,下调Thy-1、Vinmentin表达水平,从而抑制角膜纤维化。

Objective:To investigate the inhibitory effect of doxycycline(Doxy)on Transforming Growth Factor-β1(TGF-β1)-induced human corneal stromal cell fibrosis.Methods:Human corneal stromal cells were extracted from corneal stromal lens after femtosecond laser-assisted small incision lenticule extraction(SMILE),and the corneal fibrosis model was established in vitro by inducing human corneal stromal cells with TGF-β1.Control group,TGF-β1 group and Doxy group were set up.The proliferation ability of each group was detected by cell counting kit-8(CCK-8)method,the proliferation ability was detected by scratch test,and the content of hydroxyproline(HYP),a key factor in collagen synthesis,was determined by enzyme-related immunosorbent assay(ELISA).Reverse transcription-quantitative PCR(RT-qPCR)was used to detect the mRNA expression of the key factors of fibrosis Thy-1 and Vimentin,and western blotting was used to detect the protein expression levels of Thy-1 and Vimentin.Results:Compared with the control group,TGF-β1 group could promote the proliferation and migration of human corneal stromal cells,and increase the content of HYP,the expression of Thy-1 and Vimentin(all P<0.05).Compared with the TGF-β1 group,the abilities of proliferation,migration and collagen synthesis were significantly decreased in the Doxy group,and the expression of Thy-1 and Vimentin was decreased(all P<0.05).Conclusion:Doxy can inhibit the proliferation,migration and collagen synthesis of human corneal stromal cells induced by TGF-β1,and down-regulate the expression levels of Thy-1 and Vinmentin,thereby inhibiting corneal fibrosis.