| 目的：本课题以小鸡形觉剥夺性近视眼（Form-deprivation myopia, FDM）动物模型为研究对象，探索基质金属蛋白酶-2（Matrix metalloproteinase-2, MMP-2）在小鸡FDM巩膜细胞外基质（Extracellular matrix, ECM）重塑中的重要作用及转化生长因子-β1（Transforming growth factor-β1, TGF-b1）对其表达与活性的调控。
方法：课题分三部分进行研究。第一部分建立小鸡FDM、近视恢复眼（Recovery of form-deprivation myopia, RFDM）动物模型，采用组织病理切片HE染色、明胶酶谱法、逆转录-聚合酶链反应（Reverse transcriptase polymerase reaction, RT-PCR）、免疫组化等方法研究MD对被剥夺眼后极部巩膜MMP-2及其组织特异性抑制剂-2（Tissue inhibitor of matrix metalloproteinase-2, TIMP-2）表达的影响。且如上法随机选取部分小鸡制备FDM动物模型，同时往正常与FDM鸡球后注射不同浓度的基质金属蛋白酶抑制剂（Tissue inhibitor of matrix metalloproteinases, TIMPs） GM6001以抑制MMP-2酶活性，每日1次，4d、14d后观察GM6001对正常与FDM小鸡眼轴长度及屈光度变化的影响以确证MMP-2在FDM巩膜重塑中的关键作用。第二部分在第一部分基础上，体外培养正常与MD（14d）小鸡眼后极部巩膜，同时以TGF-b1、肿瘤坏死因子-α（Tumor necrosis factor-α, TNF-a）、纤维连接蛋白（Fibronectin, FN）、转铁蛋白(Ovotransferrin)、碱性成纤维细胞生长因子（basic fibroblast growth factor, bFGF）进行干预，24h后采用明胶酶谱法检测MMP-2酶活性水平变化，采用RT-PCR法检测MMP-2、TIMP-2 mRNA表达的变化以筛选出调控MMP-2表达的阳性细胞因子。第三部分采用球后注射法给正常与MD鸡眼注射尿激酶型纤溶酶原激活剂（Urokinase pasminogen activator, uPA）、纤溶酶原激活剂抑制剂-1（Plasminogen activator inhibitor-1, PAI-1）以促进或抑制TGF-β表达与活化，每日1次，14d后观察uPA、PAI-1对正常与FDM小鸡眼轴与屈光度变化的影响，同时采用与第一章相同方法检测小鸡后极部巩膜TGF-β1、MMP-2、TIMP-2表达的变化，以探讨TGF-b1对FDM后极部巩膜MMP-2表达与活性的影响及揭示TGF-b1对FDM巩膜重塑调控的可能机制。
结果：第一部分结果：⑴小鸡FDM眼后极部巩膜MMP-2酶活性、mRNA表达均显著增高，而TIMP-2降低，与正常对照组、自身对照组比较差别有统计学意义（P<0.001）。且此种改变呈明显时间依赖性，随剥夺时间延长其变化越明显，尤其是在被剥夺4-14d期间变化最明显，不同剥夺时间组组间差异有统计学意义（P<0.001）。⑵MD7d后去遮盖4d上述改变得到逆转，而MD 21后去遮盖4d上述改变仅能得到部分逆转。⑶GM6001能明显抑制MD 4d小鸡眼轴延长与近视性屈光度增加，与阴性对照组比较差别有统计学意义（P<0.001），但对MD 14d小鸡仅起部分抑制作用。GM6001对正常小鸡无明显作用。第二部分结果：TGF-β1干预的培养的正常与MD 小鸡后极部巩膜MMP-2酶活性及mRNA表达均显著降低，与阴性对照组比较差异有统计学意义（P<0.001）。且TGF-β1干预的MD小鸡后极部巩膜TIMP-2 mRNA表达增高，而对正常小鸡无明显作用。第三部分结果：⑴uPA可显著抑制正常与MD小鸡眼轴延长及近视屈光度增加，PAI-1仅可明显刺激正常小鸡眼轴延长及促使其往近视化发展(P<0.001)，而对MD小鸡无明显作用。⑵形觉剥夺能明显降低MD眼后极部巩膜TGF-β1 mRNA表达，与正常对照组、自身对照组比较差别有统计学意义（P<0.001）；uPA可促进正常与MD小鸡眼后极部巩膜TGF-β1 mRNA与蛋白表达，PAI-1可抑制正常眼TGF-b1表达，对剥夺眼无作用；⑶uPA可明显抑制正常与MD眼后极部巩膜MMP-2酶活性水平(P<0.001)，PAI-1对正常眼起上调作用，但对MD眼无影响。⑷uPA仅可轻度促进FDM眼后极部巩膜TIMP-2 mRNA与蛋白表达，而PAI-1仅抑制正常眼后极部巩膜TIMP-2表达(P<0.05)。
结论：⑴后极部巩膜MMP-2酶活性特异性增高为小鸡FDM巩膜重塑的重要直接因素，而TIMP-2抑制与基因表达转录调节参与MMP-2酶活性调节过程；⑵TGF-β1能明显抑制小鸡后极部巩膜MMP-2酶活性，其调节机理与影响MMP-2基因表达转录水平有关；⑶小鸡FDM后极部巩膜MMP-2表达特异性增高与形觉剥夺特异性抑制后极部巩膜TGF-β1表达与活化，使TGF-β1对MMP-2表达与酶活性的抑制作用得以解除密切相关。
关键词 高度近视眼，基质金属蛋白酶-2，转化生长因子-β1，巩膜重塑，小鸡
Important role of increased Matrix Metalloproteinase-2 activity regulated by transforming growth factor beta1 in the active scleral remodeling process during the development of form-deprivation myopia in chicks.
Purpose: To investigate the key role of matrix metalloproteinase-2 (MMP-2) in the active scleral remodeling process of extracellular matrix(ECM) and the effect of transforming growth factor-β1(TGF-β1) on the expression of mRNA, pro-enzyme and enzymic activity of MMP-2 during the development of form-deprivation myopia (FDM) of chicks.
Methods: The current study was divided into three parts. The first part was that: one-day-old chicks were randomly chosen to establish the animal models of FDM and recovery of form-deprivation myopia (RFDM), and one step reverse transcriptiontase-polymerase chain reaction (RT-PCR) and histochemical method were performed respectively to measure the expression of mRNA, and pro- or active protein of MMP-2 and its specific tissue inhibitor (TIMP-2). and meanwhile gelatin enzymography was also used to determined the level of activity of MMP-2 in the posterior sclera of these experimental eyes. To determine the important role of MMP-2 in the development of FDM different dose of a synthetic inhibitor of matrix metalloproteinase GM6001were daily administered into the deprived eyes to inhibit the activity of MMP-2. After four or fourteen days’treatment the effect of GM6001 on the active remodeling of ECM in the posterior sclera and the development of FDM was determined by changes of axial length and diopters of experimental chick eyes. The second part was that: on the basis of what had been attained in the first part the posterior sclera required from normal and deprived eyes were cultured, and at the same time five interference factors, that is tumor necrosic factor-α(TNF-α) (2.5ng/ml), basic fibroblast growth factor(bFGF) (10ng/ml), Ovotransferrin(500ng/ml), fibronectin (0.2mg/ml) and TGF-β1 (10ng/ml) were added into the culture medium. After 24 hours’ incubation the level of MMP-2 activity was measured by gelatin enzymography, and the levels of MMP-2 and TIMP-2 mRNA were evaluated by one step RT-PCR to chosen the postive modulation factors for MMP-2 and TIMP-2 gene expression. The last part was that: Urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) selected were administered to determined the effect of TGF-β1 activation on the modulation of MMP-2 activity and its mRNA expression, and on TIMP-2 gene expression, and on changes of the axial length and diopters in normal and in FDM eyes.
Results: The results attained from first part were as follows: ⑴compared with normal and self-controlled groups, the expression levels of MMP-2 activity, mRNA and its pro-enzyme in deprived eyes were much higher(P<0.001), and while the levels of TIMP-2 mRNA and its protein were lower(P<0.001). With increase of the time of monocular deprivation these changes were more significant and reached the highest or lowest point on 4-14th day with an intergroup statistical difference(P<0.01).⑵After removed the diffuser for 4d, changes in gene expression of MMP-2 and TIMP-2 metioned above could be recovered ultimately or partly in chicks deprived vision for 7d or 21d respectively. ⑶GM6001 could completely inhibit the development of FDM in chicks deprived vision for 4d, and while in chicks deprived for 14d its effects lessened obviously, and there was no effect on that of nondeprived chicks. What had been got from the second part was that: compared with groups treated with TNF-α, bFGF and Ovotransferrin both in the normal and MD eyes the expression levels of MMP-2 activity and pro-enzyme and its mRNA in the groups treated with TGF-β1 were lower (P<0.001). As for TIMP-2 mRNA expression levels the thing was different. In MD group treated with TGF-β1 the level of TIMP-2 mRNA increased slightly(P<0.05), and no changes were found in normal group(P>0.05). The results found in the last part were as follows: ⑴ In the non-deprived eyes, the axial length and diopter degree were reduced after uPA treatment, whereas PAI-1 increased them. In the deprived eyes, uPA inhibited axial length elongation and the development of FDM, but PAI-1 had no effect on it.⑵The contents of TGF-β1 mRNA and protein in the posterior sclera were reduced significantly in FDM eyes compared with the control specimen(P<0.001). In the non-deprived and deprived eyes, the levels of TGF-β1 mRNA and protein were increased after uPA treatment, whereas the effects of PAI-1 on them in non-deprived group was just contrary, and there was no effect was found in FDM group. ⑶In the non-deprived and deprived eyes, the levels of MMP-2 mRNA and enzymic activity and pro-enzyme were reduced after uPA treatment, whereas the effects of PAI-1 on them in non-deprived group was increased , and there was no effect was found in FDM group.⑷uPA could only slightly increase the TIMP-2 gene expression levels in deprived group, and PAI-1 could only reduced it in non-deprived group.
Conclusion:⑴That MMP-2 activity expressed significant high in the posterior sclera would be the directed key factor to trigger sclera ECM remodeling process therefore to develop FDM, in which inhibition effect of TIMP-2 and transcriptional modulation of MMP-2 gene expression would be important in modulation of MMP-2 activity.⑵Probably by influencing its transcriptional modulation of gene expression TGF-β1 could significantly inhibit the expression of MMP-2 gene in the posterior sclera in FDM;⑶Specific high level of MMP-2 activity would be resulted from elimation of the inhibition effect of TGF-β1 whose expressional level were reduced significantly during the development of FDM.
Key-words: form-deprivation myopia; matrix metalloproteinase-2; transforming growth factor-β1; scleral remodeling. |
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