吡非尼酮抑制转化生长因子β1诱导的大鼠角膜基质细胞纤维化
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摘要
目的观察吡非尼酮(PFD)对转化生长因子β1(TGF-β1)诱导的大鼠角膜基质细胞向成纤维细胞转化过程的影响,探讨PFD抗角膜基质纤维化的作用。方法分离培养大鼠角膜基质细胞,免疫细胞化学法检测波形蛋白(Vimentin)进行细胞鉴定。实验分为对照组(DMEM+10%FBS)、TGF-β1组(2 ng/mL TGF-β1+DMEM+10%FBS)和PFD组(1 mg/mL PFD+2 ng/mL TGF-β1+DMEM+10%FBS)。CCK-8法检测细胞增殖能力,Western blot检测细胞CollagenⅠ、CollagenⅢ、Keratocan和CD90蛋白表达。结果大鼠角膜基质细胞呈Vimentin胞浆阳性。与对照组及TGF-β1组比较,PFD组细胞的增殖OD值显著减低(P<0.05)。Western blot显示PFD组的细胞CollagenⅠ、Keratocan蛋白表达增强而CollagenⅢ和CD90蛋白表达减弱(P<0.05)。PFD组的CollagenⅢ/CollagenⅠ比值在3组中最低(P<0.05)。结论 PFD抗角膜基质细胞纤维化是通过抑制TGF-β分子途径,从而影响胶原合成和Keratocan、CD90蛋白表达实现的。
Objective To study the effect of pirfenidone(PFD)on the transformation of rat cornealstromal cells into fibroblasts in vitro and further explore the anti-fibrotic effect of PFD. Methods The cornealstromal cells from SD rat was isolated and cultured,and was determined by vimentin stain. The experiment wasdivided into control group(DMEM + 10%FBS),TGF-β1 group(2 ng/mL TGF-β1+ DMEM + 10%FBS)and PFDgroup(1 mg/mL PFD + 2 ng/mL TGF-β1 + DMEM + 10%FBS). Cell proliferation was detected by CCK-8 assay.Collagen Ⅰ,Collagen Ⅲ,Keratocan and CD99 expression were detected by Western blot. Results Comparedwith control group and TGF-β1 group,the cell proliferation were significantly decreased in PFD group(P < 0.05).Western blot showed that PFD can up-regulated Collagen Ⅰ and Keratocan but down-regulated Collagen Ⅲ CD90 expression(P < 0.05). The ratio of Collagen Ⅲ/Collagen Ⅰ in PFD group was lowest in all groups(P < 0.05).Conclusion PFD can resistant fibration in corneal stromal cells may through the inhibition of TGF-β,whichaffect the collagen synthesis and Keratocan,CD90 expression.
Objective To study the effect of pirfenidone(PFD)on the transformation of rat cornealstromal cells into fibroblasts in vitro and further explore the anti-fibrotic effect of PFD. Methods The cornealstromal cells from SD rat was isolated and cultured,and was determined by vimentin stain. The experiment wasdivided into control group(DMEM + 10%FBS),TGF-β1 group(2 ng/mL TGF-β1+ DMEM + 10%FBS)and PFDgroup(1 mg/mL PFD + 2 ng/mL TGF-β1 + DMEM + 10%FBS). Cell proliferation was detected by CCK-8 assay.Collagen Ⅰ,Collagen Ⅲ,Keratocan and CD99 expression were detected by Western blot. Results Comparedwith control group and TGF-β1 group,the cell proliferation were significantly decreased in PFD group(P < 0.05).Western blot showed that PFD can up-regulated Collagen Ⅰ and Keratocan but down-regulated Collagen Ⅲ CD90 expression(P < 0.05). The ratio of Collagen Ⅲ/Collagen Ⅰ in PFD group was lowest in all groups(P < 0.05).Conclusion PFD can resistant fibration in corneal stromal cells may through the inhibition of TGF-β,whichaffect the collagen synthesis and Keratocan,CD90 expression.
引文
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[13]VARKOLY G,BENCZE J,HORTOBáGYI T,et al.The cor-neal wound healing and the extracellular matrix[J].Orv Hetil,2016,157(25):995-999.
[14]LIU C Y,SHIRAISHI A,KAO C W,et al.The cloning ofmouse keratocan c DNA and genomic DNA and the characteriza-tion of its expression during eye development[J].J BiolChem,1998,273(35):22584-2258.
[15]LYNCH A P,O′SULLIVAN F,AHEARNE M.The effect ofgrowth factor supplementation on corneal stromal cell phenotypein vitro using a serum-free media[J].Exp Eye Re,2016,103(10):26-37.
[16]SIDNEY L E,BRANCH M J,DUA H S,et al.Effect of cul-ture medium on propagation and phenotype of corneal stroma-derived stem cells[J].Cytotherapy,2015,17(12):1706-1722.
[17]PEI Y,SHERRY D M,MCDERMOTT A M.Thy-1 distinguish-es human corneal fibroblasts and myofibroblasts from kerato-cytes[J].Exp Eye Res,2004,79(5):705-712.
[2]GEORGE P M,WELLS A U.Pirfenidone for the treatment ofidiopathic pulmonary fibrosis[J].Expert Rev Clin Pharmacol,2017,10(5):483-491.
[3]TZOUVELEKIS A,NTOLIOS P,KARAMPITSAKOS T,et al.Safety and efficacy of pirfenidone in severe Idiopathic Pulmo-nary Fibrosis a real-world observational study[J].Pulm Phar-macol Ther,2017,46:48-53.
[4]陈俊杰,吴共发,林俊汕,等.吡非尼酮对大鼠角膜基质细胞增殖的影响[J].国际眼科杂志,2015,15(2):201-204.
[5]TORRICELLI A A,SANTHANAM A,WU J,et al.The corne-al fibrosis response to epithelial-stromal injury[J].Exp EyeRes,2016,103(1):110-118.
[6]张露,李霞.TGF-β在角膜损伤修复中的时间和空间分布[J].眼科新进展,2017,37(2):184-188.
[7]LJUBIMOV A V,SAGHIZADEH M.Progress in corneal woundhealing[J].Prog Retin Eye Res,2015,49(6):17-45.
[8]CHOWDHURY S,GUHA R,TRIVEDI R,et al.Pirfenidonenanoparticles improve corneal wound healing and prevent scar-ring following alkali burn[J].PLo S One,2013,8(8):e70528.
[9]FINK M K,GIULIANO E A,TANDON A,et al.Therapeuticpotential of Pirfenidone for treating equine corneal scarring[J].Vet Ophthalmol,2015,18(3):242-250.
[10]ETHEREDGE L,KANE B P,HASSELL J R.The effect ofgrowth factor signaling on keratocytes in vitro and its relation-ship to the phases of stromal wound repair[J].Invest Ophthal-mol Vis Sci,2009,50(7):3128-3136.
[11]方素芬,郭武华.类泛素缀和酶9在肝纤维化中的作用及机制[J].实用医学杂志,2017,33(13):2079-2082.
[12]BOEHME S A,FRANZ-BACON K,DITIRRO D N,et al.MAP3K19 is a novel regulator of TGF-βsignaling that impactsbleomycin-induced lung injury and pulmonary fibrosis[J].PLo S One,2016,11(5):e0154874.
[13]VARKOLY G,BENCZE J,HORTOBáGYI T,et al.The cor-neal wound healing and the extracellular matrix[J].Orv Hetil,2016,157(25):995-999.
[14]LIU C Y,SHIRAISHI A,KAO C W,et al.The cloning ofmouse keratocan c DNA and genomic DNA and the characteriza-tion of its expression during eye development[J].J BiolChem,1998,273(35):22584-2258.
[15]LYNCH A P,O′SULLIVAN F,AHEARNE M.The effect ofgrowth factor supplementation on corneal stromal cell phenotypein vitro using a serum-free media[J].Exp Eye Re,2016,103(10):26-37.
[16]SIDNEY L E,BRANCH M J,DUA H S,et al.Effect of cul-ture medium on propagation and phenotype of corneal stroma-derived stem cells[J].Cytotherapy,2015,17(12):1706-1722.
[17]PEI Y,SHERRY D M,MCDERMOTT A M.Thy-1 distinguish-es human corneal fibroblasts and myofibroblasts from kerato-cytes[J].Exp Eye Res,2004,79(5):705-712.