机械牵拉与前列腺素E_2联合作用下调圆锥角膜成纤维细胞赖氨酰氧化酶家族基因表达
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摘要
为了研究机械牵拉与前列腺素E_2(PGE_2)对赖氨酰氧化酶(LOXs)家族基因表达的影响,本文对体外培养的正常人及圆锥角膜患者角膜成纤维细胞给予PGE_2处理,并进行幅度为12%、频率为0.1 Hz的周期性机械牵拉12 h,采用荧光定量聚合酶链式反应(PCR)检测LOXs家族基因的表达情况。研究结果显示,圆锥角膜患者的角膜成纤维细胞LOXs家族基因表达量普遍低于正常角膜;与静态培养组比较,单独机械牵拉使正常组的赖氨酰氧化酶样蛋白(LOXL)中的LOXL-2和LOXL-4基因表达上调,并使得圆锥角膜组的LOXL-3和LOXL-4基因表达下调;机械牵拉与PGE_2联合作用则使正常组的LOXL-4以及圆锥角膜组除LOXL-1之外的所有LOXs基因表达均下调。根据以上研究结果可提示,以机械牵拉和PGE_2同时作用,将下调圆锥角膜患者角膜成纤维细胞的LOXs家族基因表达,这将可能导致角膜胶原交联受阻,使胶原纤维之间的黏附力下降,胶原板层之间产生相对滑动,影响角膜结构稳定性,促进角膜发生圆锥膨隆。本文通过探讨研究力学刺激与炎性因子对LOXs家族基因表达的影响,有助于更好地理解圆锥角膜发生发展的可能机理,为圆锥角膜预防及治疗提供参考。
In order to investigate the effects of mechanical stretching combined with prostaglandin E_2(PGE_2) on the gene expression of lysyl oxidases(LOXs) in keratoconus, we treated cultured corneal fibroblasts from healthy human cornea and keratoconus patient cornea with PGE_2 and/or cyclic stretch(12% elongation, 0.1 Hz, 12 h). Real-time fluorescent quantitative polymerase chain reaction was used to detect the gene expression of LOXs. The results showed that the gene expression of LOXs in keratoconus group was significantly lower than that in the healthy one. Compared to the static control group, 12% stretching alone up-regulated gene expression of LOXL-2, LOXL-4 in the healthy group,whereas it down-regulated LOXL-3, LOXL-4 in the keratoconus group. Combination of 12% stretching and PEG_2 induced LOXL-4 down-regulation in in healthy group, and all LOXs except LOXL-1 in keratoconus group. The results suggested that combination of mechanical stretching and PGE_2 down-regulate the gene expression of LOXs in keratoconus. Lower LOXs expression may lead to impaired cross-linking, and thus to a loss of cohesion between collagen fibrils, affecting corneal structural stability by collagen lamellae slippage. This may facilitate the development of keratoconus. Exploring the effects of mechanical stretching and inflammatory factor on the expression LOXs in this paper will help us to understand the possible mechanism of how the keratoconus occurs and develops well, and provide the reference for the prevention and treatment of keratoconus.
In order to investigate the effects of mechanical stretching combined with prostaglandin E_2(PGE_2) on the gene expression of lysyl oxidases(LOXs) in keratoconus, we treated cultured corneal fibroblasts from healthy human cornea and keratoconus patient cornea with PGE_2 and/or cyclic stretch(12% elongation, 0.1 Hz, 12 h). Real-time fluorescent quantitative polymerase chain reaction was used to detect the gene expression of LOXs. The results showed that the gene expression of LOXs in keratoconus group was significantly lower than that in the healthy one. Compared to the static control group, 12% stretching alone up-regulated gene expression of LOXL-2, LOXL-4 in the healthy group,whereas it down-regulated LOXL-3, LOXL-4 in the keratoconus group. Combination of 12% stretching and PEG_2 induced LOXL-4 down-regulation in in healthy group, and all LOXs except LOXL-1 in keratoconus group. The results suggested that combination of mechanical stretching and PGE_2 down-regulate the gene expression of LOXs in keratoconus. Lower LOXs expression may lead to impaired cross-linking, and thus to a loss of cohesion between collagen fibrils, affecting corneal structural stability by collagen lamellae slippage. This may facilitate the development of keratoconus. Exploring the effects of mechanical stretching and inflammatory factor on the expression LOXs in this paper will help us to understand the possible mechanism of how the keratoconus occurs and develops well, and provide the reference for the prevention and treatment of keratoconus.
引文
1 Gordon-Shaag A,Millodot M,Shneor E,et al.The genetic and environmental factors for keratoconus.Biomed Res Int,2015,795738:1-19.
2 Chen Xi,Greenaway F T.Identification of the disulfide bonds of lysyl oxidase.J Neural Transm,2011,118(7,SI):1111-1114.
3胡玲玲,汤其群,黄海艳.赖氨酰氧化酶的结构和功能.生命的化学,2009,29(2):291-294.
4 Akhtar S,Bron A J,Salvi S M,et al.Ultrastructural analysis of collagen fibrils and proteoglycans in keratoconus.Acta Ophthalmol,2008,86(7):764-772.
5 Shore R C,Moxham B J,Berkovitz B B.Ultrastructural analysis of collagen fibrils and proteoglycans in keratoconus.Connect Tissue Res,1984,12(3/4):249-255.
6 Farzaneh H L,Ramin S,Validad M H,Association of Lysyl oxidase(LOX)polymorphisms with the risk of keratoconus in an iranian population.Association of Lysyl oxidase(LOX)polymorphisms with the risk of keratoconus in an iranian population.Ophthalmic Genet,2015,36(4):309-314.
7 Dudakova L,Liskova P,Trojek T,et al.Changes in lysyl oxidase(lox)distribution and its decreased activity in keratoconus corneas.Exp Eye Res,2012,104(8):74-81.
8 Dudakova L,Sasaki T,Liskova P,et al.The presence of lysyl oxidase-like enzymes in human control and keratoconic corneas.Histol Histopathol,2016,31(1):63-71.
9 Shetty R,Sathyanarayanamoorthy A,Ramachandra R A,et al.Attenuation of lysyl oxidase and collagen gene expression in keratoconus patient corneal epithelium corresponds to disease severity.Mol Vis,2015,21:12-25.
10刘成星,冯鹏飞,李晓娜,等.机械牵张对兔角膜成纤维细胞细胞外基质基因表达的影响.医用生物力学,2014,29(5):447-453,458.
11 Liu Chengxing,Feng Pengfei,Li Xiaona,et al.Expression of MMP-2,MT1-MMP,and TIMP-2 by cultured rabbit corneal fibroblasts under mechanical stretch.Exp Biol Med,2014,239(8):907-912.
12 FENG Pengfei,LI Xiaona,CHEN Weiyi,et al.Combined effects of interleukin-1βand cyclic stretching on metalloproteinase expression in corneal fibroblasts in vitro.Biomed Eng Online,2016,15(1):1-10.
13 Wisse R P,Kuiper J J,Gans R,et al.Cytokine expression in keratoconus and its corneal microenvironment:a systematic review.Ocul Surf,2015,13(4):272-283.
14 Mcmonnies C W.Inflammation and keratoconus.Optom Vis Sci,2015,92(2):e35-e41.
15 Bureau J,Fabre E J,Hecquet C,et al.Modification of prostaglandin-e2 and collagen-synthesis in keratoconus fibroblasts,associated with an increase of interleukin-1-alpha receptor number.C R Acad SciⅢ,1993,316(4):425-430.
16 Voloshenyuk T G,Hart A D,Khoutorova E A.TNF-alpha increases cardiac fibroblast lysyl oxidase expression through TGF-βand PI3Kinase signaling pathways.Biochem Biophys Res Commun,2011,413(2):370-375.
17 Boak A M,Roy R,Berk J,et al.Regulation of lysyl oxidase expression in lung fibroblasts by transforming growth factor-beta 1and prostaglandin E2.Am J Respir Cell Mol Biol,1994,11(6):751-755.
18 Liu Chao,Wang Wangsheng,Sun Gang.Effects of prostaglandin E2 on lysyl oxidase-mediated collagen cross-linking in human amnion fibroblasts.Placenta,2014,35(9):A44-A45.
19陈荣富.应力损伤和PGE2对共培养的PCL细胞和滑膜细胞中LOX家族及MMP-1、2、3表达的影响.重庆:重庆医科大学,2012.
20 Xie Jing,Jiang Jiahuan,Zhang Yanjun,et al.Up-regulation expressions of lysyl oxidase family in Anterior Cruciate Ligament and Medial Collateral Ligament fibroblasts induced by Transforming Growth Factor-Beta 1.Int Orthop,2012,36(1):207-213.
21 Micheal S,Khan M I,Akhtar F,et al.Role of lysyl oxidaselike 1gene polymorphisms in Pakistani patients with pseudoexfoliative glaucoma.Mol Vis,2012,18:1040-1044.
22 Coral K,Angayarkanni N,Madhavan J,et al.Lysyl oxidase activity in the ocular tissues and the role of LOX in proliferative diabetic retinopathy and rhegmatogenous retinal detachment.Invest Ophthalmol Vis Sci,2008,49(11):4746-4752.
23 Dudakova L,Jirsova K.The impairment of lysyl oxidase in keratoconus and in keratoconus-associated disorders.J Neural Transm,2013,120(6,SI):977-982.
24 Du G,Liu C,Li X,et al.Induction of matrix metalloproteinases-1by tumor necrosis factor-αis mediated by interleukin-6 in cultured fibroblasts of keratoconus.Exp Bio Med,2016,241(18):2033-2041.
2 Chen Xi,Greenaway F T.Identification of the disulfide bonds of lysyl oxidase.J Neural Transm,2011,118(7,SI):1111-1114.
3胡玲玲,汤其群,黄海艳.赖氨酰氧化酶的结构和功能.生命的化学,2009,29(2):291-294.
4 Akhtar S,Bron A J,Salvi S M,et al.Ultrastructural analysis of collagen fibrils and proteoglycans in keratoconus.Acta Ophthalmol,2008,86(7):764-772.
5 Shore R C,Moxham B J,Berkovitz B B.Ultrastructural analysis of collagen fibrils and proteoglycans in keratoconus.Connect Tissue Res,1984,12(3/4):249-255.
6 Farzaneh H L,Ramin S,Validad M H,Association of Lysyl oxidase(LOX)polymorphisms with the risk of keratoconus in an iranian population.Association of Lysyl oxidase(LOX)polymorphisms with the risk of keratoconus in an iranian population.Ophthalmic Genet,2015,36(4):309-314.
7 Dudakova L,Liskova P,Trojek T,et al.Changes in lysyl oxidase(lox)distribution and its decreased activity in keratoconus corneas.Exp Eye Res,2012,104(8):74-81.
8 Dudakova L,Sasaki T,Liskova P,et al.The presence of lysyl oxidase-like enzymes in human control and keratoconic corneas.Histol Histopathol,2016,31(1):63-71.
9 Shetty R,Sathyanarayanamoorthy A,Ramachandra R A,et al.Attenuation of lysyl oxidase and collagen gene expression in keratoconus patient corneal epithelium corresponds to disease severity.Mol Vis,2015,21:12-25.
10刘成星,冯鹏飞,李晓娜,等.机械牵张对兔角膜成纤维细胞细胞外基质基因表达的影响.医用生物力学,2014,29(5):447-453,458.
11 Liu Chengxing,Feng Pengfei,Li Xiaona,et al.Expression of MMP-2,MT1-MMP,and TIMP-2 by cultured rabbit corneal fibroblasts under mechanical stretch.Exp Biol Med,2014,239(8):907-912.
12 FENG Pengfei,LI Xiaona,CHEN Weiyi,et al.Combined effects of interleukin-1βand cyclic stretching on metalloproteinase expression in corneal fibroblasts in vitro.Biomed Eng Online,2016,15(1):1-10.
13 Wisse R P,Kuiper J J,Gans R,et al.Cytokine expression in keratoconus and its corneal microenvironment:a systematic review.Ocul Surf,2015,13(4):272-283.
14 Mcmonnies C W.Inflammation and keratoconus.Optom Vis Sci,2015,92(2):e35-e41.
15 Bureau J,Fabre E J,Hecquet C,et al.Modification of prostaglandin-e2 and collagen-synthesis in keratoconus fibroblasts,associated with an increase of interleukin-1-alpha receptor number.C R Acad SciⅢ,1993,316(4):425-430.
16 Voloshenyuk T G,Hart A D,Khoutorova E A.TNF-alpha increases cardiac fibroblast lysyl oxidase expression through TGF-βand PI3Kinase signaling pathways.Biochem Biophys Res Commun,2011,413(2):370-375.
17 Boak A M,Roy R,Berk J,et al.Regulation of lysyl oxidase expression in lung fibroblasts by transforming growth factor-beta 1and prostaglandin E2.Am J Respir Cell Mol Biol,1994,11(6):751-755.
18 Liu Chao,Wang Wangsheng,Sun Gang.Effects of prostaglandin E2 on lysyl oxidase-mediated collagen cross-linking in human amnion fibroblasts.Placenta,2014,35(9):A44-A45.
19陈荣富.应力损伤和PGE2对共培养的PCL细胞和滑膜细胞中LOX家族及MMP-1、2、3表达的影响.重庆:重庆医科大学,2012.
20 Xie Jing,Jiang Jiahuan,Zhang Yanjun,et al.Up-regulation expressions of lysyl oxidase family in Anterior Cruciate Ligament and Medial Collateral Ligament fibroblasts induced by Transforming Growth Factor-Beta 1.Int Orthop,2012,36(1):207-213.
21 Micheal S,Khan M I,Akhtar F,et al.Role of lysyl oxidaselike 1gene polymorphisms in Pakistani patients with pseudoexfoliative glaucoma.Mol Vis,2012,18:1040-1044.
22 Coral K,Angayarkanni N,Madhavan J,et al.Lysyl oxidase activity in the ocular tissues and the role of LOX in proliferative diabetic retinopathy and rhegmatogenous retinal detachment.Invest Ophthalmol Vis Sci,2008,49(11):4746-4752.
23 Dudakova L,Jirsova K.The impairment of lysyl oxidase in keratoconus and in keratoconus-associated disorders.J Neural Transm,2013,120(6,SI):977-982.
24 Du G,Liu C,Li X,et al.Induction of matrix metalloproteinases-1by tumor necrosis factor-αis mediated by interleukin-6 in cultured fibroblasts of keratoconus.Exp Bio Med,2016,241(18):2033-2041.