MMP-2在长波光诱导新生豚鼠近视化模型巩膜胶原代谢中的作用机制研究
摘要
近视,是指眼球屈光力和眼球轴长不能精确配合导致视像聚焦于视网膜前,伴随眼球前后径延长的现象。包括人类在内的哺乳动物,在视觉发育的关键时期,改变视觉体验,将影响原有的正视化进程,导致屈光不正。由于色像差的存在,长波光成焦点于正视眼视网膜后方,本研究目的是观察长波光对新生豚鼠眼球屈光状态的影响并初步探讨基质金属蛋白酶-2(MMP-2)在长波光诱导新生豚鼠近视化模型巩膜胶原代谢中的作用机制。
本研究分三部分进行:(一)长波光诱导近视化模型的建立。采用新生豚鼠作为实验动物,观察长波光照射和混和光照射2周、4周及经过4周长波光照射后去除单纯长波光照射的恢复期豚鼠随时间推移所造成的屈光度和眼球参数的变化趋势,从而了解长波光照射对眼球屈光状态和发育的影响。经分析发现:长波光照射较混合光照射使新生豚鼠更易获得近视伴有眼轴延长主要是玻璃体腔深度的增加,其差别在实验2周时不明显,而在实验4周时显著,去除单纯长波光照射2周可以缩小这一差别,延缓长波光照射组的近视化趋势;此外,经相关分析,实验4周时各实验眼玻璃体腔深度增加量和所获得屈光度呈明显的正相关性。本部分实验成功建立长波光诱导新生豚鼠近视化模型,证明在眼球发育的早期阶段,长波光照射可诱导豚鼠近视化发生。(二)观察长波光诱导新生豚鼠近视化模型巩膜胶原代谢的改变,探讨长波光诱导新生豚鼠近视化的分子水平发生机制。通过检测长波光照射组,混合光照射组和正常对照组前、后部巩膜实验2周、实验4周和实验6周巩膜胶原密度(胶原特殊染色)和Ⅰ型胶原表达(免疫组化方法)探讨巩膜胶原的改变。经分析发现,在长波光诱导新生豚鼠近视化过程中,胶原密度减少,Ⅰ型胶原表达降低;而在去除诱导因素后则使变化向相反方向发展,表现为巩膜胶原密度增加,Ⅰ型胶原表达升高,这种区别(长波光照射组和混合光照射组)在部位上只存在于后部巩膜;时间上(实验2周)早于屈光度和眼轴发生变化的时间(实验4周)。本部分研究证明在长波光诱导新生豚鼠近视化过程中存在胶原代谢的改变,表现为后部巩膜胶原密度降低,在长波光诱导近视化早期出现。(三)探讨与近视发生明显相关的胶原降解酶MMP-2在长波光诱导新生豚鼠近视化模型巩膜胶原代谢中的作用机制。(1)通过检测长波光照射组,混合光照射组和正常对照组前、后部巩膜实验2周、实验4周和实验6周巩膜MMP-2的活性、总量及分布并进行对比分析。活性检测采用明胶酶谱法,
总量检测采用western-blot(蛋白水平)和RT-PCR(mRNA水平),形态学分布检测采用免疫组化方法;除MMP-2外,同时检测基质金属蛋白酶组织抑制剂-2(TIMP-2)和转化生长因子β(TGF-β)的蛋白表达变化(western-blot)以探讨MMP-2的活性调控。经分析发现,在长波光诱导近视化过程中,后部巩膜MMP-2活性增加,包括酶的活性成分增加和酶总量增加;蛋白和核酸水平变化一致;时间上(光照2周)与胶原变化相一致;TIMP-2蛋白表达变化趋势与MMP-2相反,而TGF-β表达变化趋势与MMP-2相同,因此推测:在长波光照射下,巩膜MMP-2活性增加,使巩膜胶原降解增加,TIMP-2和TGF-β对MMP-2活性可能分别起到抑制和促进的作用。(2)通过引入MMP-2特异性抑制剂1-(12-羟)18烷基硫酸钠抑制MMP-2活性后观察长波光对新生豚鼠近视诱导作用的变化。经分析发现,MMP-2抑制剂使长波光对新生豚鼠近视化的诱导作用减弱,呈剂量-效应依赖关系,由此进一步说明MMP-2在长波光照射诱导新生豚鼠近视化过程中起重要作用。
综合三部分研究,可以推论,在视觉发育早期,长波光照射可诱导新生豚鼠近视化,可能的机制是:长波光照射启动某一种或几种机制作用于新生豚鼠巩膜的MMP-2,使其活性增加,降解胶原的能力增加,导致后部巩膜胶原减少,抗拉伸力减弱,在眼内压的作用下易于延伸,使眼轴延长,发生近视。本课题从长波光照射下导致巩膜组织重塑的角度证明视觉发育的可塑性,并初步探讨MMP-2在其中的作用机制,为近视的发病机制和治疗方法的研究开辟新的思路。
Myopia is a common form of ocular pathlolgy in which the visual image is focused anterior to the retina resulting in an increase in the anterior-posterior(axial)dimension of the eye and is associated with extracellular matrix remodeling caused by the increased activity of MMP-2 in the sclera.A normal visually guided emmetropization mechanism fine tunes the growth of the juvenile eye to match the eye’s axial length to its optical power.Because the fact that long wavelength light is focuced behind the retina,as an abnormal visual experience, long wavelength light irridation may affect the normal process of emmetropization and cause myopia.The purpose of this study is to investigate the effect of long wavelength light to refractive status and the role of MMP-2 in scleral collagen metabolism of myopia induced by long wavelength light irradiation.
This study included three parts: 1) Construction of an animal model of myopia by raising Guinea pigs in long wavelength light irridation,optical and structural parameters were measured to prove it.The results showed that long wavelength light exposure group was more myopic than the control group, which were raised under white light and natural raise condition(P<0.001), and the axial length elongation especially the vitreous chamber depth elongation of treated group were statistically longer than that of control groups(P<0.001) and the gained myopia diopter was significantly correlated with the vitreous chamber depth elongation in treated group(r=0.803,P=0.002).2)To investigate the mechanism of this phenomenon, we chosed the sclera as target tissue and detected the effects of long wavelength light on the collagen metabolism of sclera,The results showed that the collagen density and the expression of collagenⅠat the posterior sclera of the long wavelength light irradiated eyes were significantly lower than that of control eyes and
normal eyes (P<0.001).3) To investigate the role of MMP-2 in scleral collagen metabolism of myopia induced by long wavelength light irradiation,the activity and the expression of MMP-2,TIMP-2,TGF-β and also the MMP-2 expression under using of synthetic specific MMP-2 inhibitor were detected. The results showed that the activity of MMP-2 and the expression of MMP-2 and TGF-β at the posterior sclera of the long wavelength light exposure eyes were significantly higher than that of control group,while the expression of TIMP-2 was lower than that of control group,synthetic specific MMP-2 inhibitor reduced the myopia induced by long wavelength light exposure through inhibiting the activity of MMP-2 in a dose-dependent manner.
So, we have a conclusion that as an abnormal visual experience, long wavelength light irridation can effect the normal process of emmetropization and cause myopia, one of its mechanism may be break the balance between the synthesis and decompose of collagen metabolism of sclera,increased MMP-2 activity plays an important role in the development of myopia induced by long wavelength light irridation. This study settled a background for the clarification of the pathogeny of myopia and indicated a new possible method of myopic therapy.
本研究分三部分进行:(一)长波光诱导近视化模型的建立。采用新生豚鼠作为实验动物,观察长波光照射和混和光照射2周、4周及经过4周长波光照射后去除单纯长波光照射的恢复期豚鼠随时间推移所造成的屈光度和眼球参数的变化趋势,从而了解长波光照射对眼球屈光状态和发育的影响。经分析发现:长波光照射较混合光照射使新生豚鼠更易获得近视伴有眼轴延长主要是玻璃体腔深度的增加,其差别在实验2周时不明显,而在实验4周时显著,去除单纯长波光照射2周可以缩小这一差别,延缓长波光照射组的近视化趋势;此外,经相关分析,实验4周时各实验眼玻璃体腔深度增加量和所获得屈光度呈明显的正相关性。本部分实验成功建立长波光诱导新生豚鼠近视化模型,证明在眼球发育的早期阶段,长波光照射可诱导豚鼠近视化发生。(二)观察长波光诱导新生豚鼠近视化模型巩膜胶原代谢的改变,探讨长波光诱导新生豚鼠近视化的分子水平发生机制。通过检测长波光照射组,混合光照射组和正常对照组前、后部巩膜实验2周、实验4周和实验6周巩膜胶原密度(胶原特殊染色)和Ⅰ型胶原表达(免疫组化方法)探讨巩膜胶原的改变。经分析发现,在长波光诱导新生豚鼠近视化过程中,胶原密度减少,Ⅰ型胶原表达降低;而在去除诱导因素后则使变化向相反方向发展,表现为巩膜胶原密度增加,Ⅰ型胶原表达升高,这种区别(长波光照射组和混合光照射组)在部位上只存在于后部巩膜;时间上(实验2周)早于屈光度和眼轴发生变化的时间(实验4周)。本部分研究证明在长波光诱导新生豚鼠近视化过程中存在胶原代谢的改变,表现为后部巩膜胶原密度降低,在长波光诱导近视化早期出现。(三)探讨与近视发生明显相关的胶原降解酶MMP-2在长波光诱导新生豚鼠近视化模型巩膜胶原代谢中的作用机制。(1)通过检测长波光照射组,混合光照射组和正常对照组前、后部巩膜实验2周、实验4周和实验6周巩膜MMP-2的活性、总量及分布并进行对比分析。活性检测采用明胶酶谱法,
总量检测采用western-blot(蛋白水平)和RT-PCR(mRNA水平),形态学分布检测采用免疫组化方法;除MMP-2外,同时检测基质金属蛋白酶组织抑制剂-2(TIMP-2)和转化生长因子β(TGF-β)的蛋白表达变化(western-blot)以探讨MMP-2的活性调控。经分析发现,在长波光诱导近视化过程中,后部巩膜MMP-2活性增加,包括酶的活性成分增加和酶总量增加;蛋白和核酸水平变化一致;时间上(光照2周)与胶原变化相一致;TIMP-2蛋白表达变化趋势与MMP-2相反,而TGF-β表达变化趋势与MMP-2相同,因此推测:在长波光照射下,巩膜MMP-2活性增加,使巩膜胶原降解增加,TIMP-2和TGF-β对MMP-2活性可能分别起到抑制和促进的作用。(2)通过引入MMP-2特异性抑制剂1-(12-羟)18烷基硫酸钠抑制MMP-2活性后观察长波光对新生豚鼠近视诱导作用的变化。经分析发现,MMP-2抑制剂使长波光对新生豚鼠近视化的诱导作用减弱,呈剂量-效应依赖关系,由此进一步说明MMP-2在长波光照射诱导新生豚鼠近视化过程中起重要作用。
综合三部分研究,可以推论,在视觉发育早期,长波光照射可诱导新生豚鼠近视化,可能的机制是:长波光照射启动某一种或几种机制作用于新生豚鼠巩膜的MMP-2,使其活性增加,降解胶原的能力增加,导致后部巩膜胶原减少,抗拉伸力减弱,在眼内压的作用下易于延伸,使眼轴延长,发生近视。本课题从长波光照射下导致巩膜组织重塑的角度证明视觉发育的可塑性,并初步探讨MMP-2在其中的作用机制,为近视的发病机制和治疗方法的研究开辟新的思路。
Myopia is a common form of ocular pathlolgy in which the visual image is focused anterior to the retina resulting in an increase in the anterior-posterior(axial)dimension of the eye and is associated with extracellular matrix remodeling caused by the increased activity of MMP-2 in the sclera.A normal visually guided emmetropization mechanism fine tunes the growth of the juvenile eye to match the eye’s axial length to its optical power.Because the fact that long wavelength light is focuced behind the retina,as an abnormal visual experience, long wavelength light irridation may affect the normal process of emmetropization and cause myopia.The purpose of this study is to investigate the effect of long wavelength light to refractive status and the role of MMP-2 in scleral collagen metabolism of myopia induced by long wavelength light irradiation.
This study included three parts: 1) Construction of an animal model of myopia by raising Guinea pigs in long wavelength light irridation,optical and structural parameters were measured to prove it.The results showed that long wavelength light exposure group was more myopic than the control group, which were raised under white light and natural raise condition(P<0.001), and the axial length elongation especially the vitreous chamber depth elongation of treated group were statistically longer than that of control groups(P<0.001) and the gained myopia diopter was significantly correlated with the vitreous chamber depth elongation in treated group(r=0.803,P=0.002).2)To investigate the mechanism of this phenomenon, we chosed the sclera as target tissue and detected the effects of long wavelength light on the collagen metabolism of sclera,The results showed that the collagen density and the expression of collagenⅠat the posterior sclera of the long wavelength light irradiated eyes were significantly lower than that of control eyes and
normal eyes (P<0.001).3) To investigate the role of MMP-2 in scleral collagen metabolism of myopia induced by long wavelength light irradiation,the activity and the expression of MMP-2,TIMP-2,TGF-β and also the MMP-2 expression under using of synthetic specific MMP-2 inhibitor were detected. The results showed that the activity of MMP-2 and the expression of MMP-2 and TGF-β at the posterior sclera of the long wavelength light exposure eyes were significantly higher than that of control group,while the expression of TIMP-2 was lower than that of control group,synthetic specific MMP-2 inhibitor reduced the myopia induced by long wavelength light exposure through inhibiting the activity of MMP-2 in a dose-dependent manner.
So, we have a conclusion that as an abnormal visual experience, long wavelength light irridation can effect the normal process of emmetropization and cause myopia, one of its mechanism may be break the balance between the synthesis and decompose of collagen metabolism of sclera,increased MMP-2 activity plays an important role in the development of myopia induced by long wavelength light irridation. This study settled a background for the clarification of the pathogeny of myopia and indicated a new possible method of myopic therapy.
引文
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44.Rada JA, Brenza HL.Increased latent gelatinase activity in the sclera of visually deprived chicks. Invest Ophthalmol Vis Sci. 1995; 36: 1555-1565.
45.Kayano K, Shimada T, Shinomiya T,et al.Activation of pro-MMP-2 mediated by MT1-MMP in human salivary gland carcinomas: possible regulation of pro-MMP-2 activation by TIMP-2. J Pathol. 2004 ;202:403-411.
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49.Martelli-Junior H, Cotrim P,Graner E,Sauk JJ,Coletta RD.Effect of
transforming growth factor-beta1,interleukin-6,and interferon-gamma on the expression of type I collagen,heat shock protein 47, matrix metalloproteinase (MMP)-1 and MMP-2 by fibroblasts from normal gingiva and hereditary gingival fibromatosis.J Periodontol.2003;74:296-306.
50.Cheng S, Lovett DH.Gelatinase A (MMP-2) is necessary and sufficient for renal tubular cell epithelial-mesenchymal transformation.Am J Pathol. 2003;162:1937-1949.
51.Asanuma K,Shirato I,Ishidoh K,Kominami E,Tomino Y.Selective modulation of the secretion of proteinases and their inhibitors by growth factors in cultured differentiated podocytes. Kidney Int.2002; 62:822-831.
52.lam DS,Lee WS Leung YF,et al.TGFbeta-induced factor:a candidate gene for high myopia.Invest Ophthalmol Vis Sci.2003;44:1012-1015.
53.Seko Y,Tanaka Y,Tokoro T.Influence of bFGF as a potent growth stimulator and TGF-beta as a growth regulator on scleral chondrocytes and scleral fibroblasts in vitro.Ophthalmic Res.1995;27:144-152.
54.Seko Y,Shimokawa H,Tokoro T.Expression of bFGF and TGF-beta 2 in experimental myopia in chicks.Invest Ophthalmol Vis Sci. 1995; 36: 1183-1187.
55.Kusakari T,Sato T,Tokoro T.Visual deprivation stimulates the exchange of the fibrous sclera into the cartilaginous sclera in chicks.Exp Eye Res.2001;73:533-546.
56.Honda S,Fujii S,Sekiya Y,et al.Retinal control on the axial length mediated by transforming growth factor-beta in chick eye.Invest Ophthalmol Vis Sci.1996;37:2519-2526.
57.Seko Y,Tanaka Y,Tokoro T.Influence of bFGF as a potent growth stimulator and TGF-beta as a growth regulator on scleral chondrocytes and scleral fibroblasts in vitro.Ophthalmic Res.1995;27:144-152.
58.Shozo FB, Kyoka T, Kazuo K, et al. Tumor detection using 18F-labeled matrix metalloproteinase -2 inhibitor.Nuclear Medicine and Biology.2003; 30:119–125.
59.Furumoto, R. Iwata, T.Design and synthesis of fluorine-18 labeled matrix metalloproteinase inhibitors for cancer imaging, J.Labelled Compd. Radiopharm.2002;45:975–986.
60.Bramhall SR,Rosemurgy A., Bowry BC,et al.Marimastat Pancreatic Cancer Study Group,Marimastat as first-line therapy for patients with unresectable pancreatic cancer:a randomized trial. J.Clin.Oncol. 2001;19: 3447–3455.
61.Fujita M,Nakao Y,Matsunaga S,et al.Sodium 1-(12-Hydroxy)octadecanyl sulfata,an MMP2 inhibitor,isolated from a tunicate of the family polyclinidae.J Nat.Prod.2002;65:1936-1938.