尿激酶型纤溶酶原激活剂诱导巩膜内源性TGF-β1表达抑制小鸡FDM形成

Effects of inner transforming growth factor-beta1 in the posterior sclera induced by urokinase pasminogen activator on the development of chick form-deprivation myopia

目的:研究外源性尿激酶型纤溶酶原激活剂(uPA)对小鸡形觉剥夺性近视眼(FDM)形成的影响及其可能的分子机制。方法:随机选取1d龄来亨雏鸡100只单眼遮盖(MD)制备FDM动物模型,同时球后注射uPA500U(50μL)、PAI-150U(50μL)或PBS50μL进行干预,1次/d。阴性对照组仅单纯遮盖。另随机选1d龄来亨雏鸡100只进行上述相同干预处理作为各自对照组。上述各组每组小鸡25只。实验前、14d后每组随机抽取10只用A超及带状视网膜检影镜测量眼轴长度及屈光度变化。摘除眼球,取后极部巩膜,HE染色法行组织病理学观察,免疫组化法检测后极部巩膜TGF-β1表达,逆转录聚合酶链反应(RT-PCR)一步法检测TGF-β1mR-NA表达变化。结果:uPA可明显抑制MD眼轴延长及近视性屈光度增加,与阴性对照组、PBS组比较差别有统计学意义(P<0.01),而PAI-1无作用;uPA可抑制正常小鸡眼轴延长及远视屈光度降低(P<0.05),而PAI-1作用正好相反,具明显促进作用(P<0.01)。正常小鸡后极部巩膜均可检测到较丰富的TGF-β1阳性表达,而FDM明显降低。uPA干预后TGF-β1阳性表达信号显著增加,而PAI-1干预后表达显著减弱。单纯MD眼后极部巩膜TGF-β1mRNA表达较正常对照组显著降低(P<0.01)。与阴性对照组、PBS组比较,uPA可上调MD与正常眼TGF-β1mRNA表达,而PAI-1对正常小鸡起下调作用(P<0.01),对MD眼无作用。结论:uPA可显著抑制小鸡FDM形成,其作用机制可能与诱导后极部巩膜内源性TGF-β1表达与活化密切相关。

AIM： To clarify whether changes of inner transforming growth factor-beta1 （TGF-β1） expression on posterior sclera, induced by urokinase pasminogen activator （uPA） or plasminogen activator inhibitor-1 （PAI-1） administrated retra-orbicularly, is the key step for the development of chick form-deprivation myopia （FDM）. METHODS： One hundred one-day-old Lethorn chicks chosen randomly were covered monocularly with plastic goggles to induce the animal model of FDM, and at the same time various interference treatments were performed. According to the variety of interference the experimental animals were divided into uPA group, PAI-1 group, PBS group and negative control group. In the former three groups uPA （500U/50μL）, PAI-1 （50U/50μL） and sterile PBS （50μL） were injected retraorbitally and respectively, and meanwhile chicks only monocularly covered were served as the negative control. Another 100 one-day-old Lethbrn chicks treated the same as the above were as normal controls. Each experimental group included 25 chicks. Before and after the 14-day experiment, 10 chicks were chosen randomly from each group and their axial length and diopter degrees were measured by A ultrasonography and streak retinoscopy, respectively. After that, the chicks were killed to acquire the posterior sclera. HE dying was performed to observe histological specialty, and the change in TGF-betal in each experiment groups was measured by one-step reverse transcriptase polymerase chain reaction （RT-PCR） and immunohistochemistry. RESULTS： Form deprivation could make the axial length of covered eye extended excessively and resulted in high myopia accordingly, in which magnificent difference between FDM group and the PBS group or its negative control group existed （P〈0.01）. Daily administration of uPA 500U/50μL could not only inhibit the development of FDM, growth of axial length and reduce of hypopic degree in normal non-covered eyes also be postponed, but also in normal control group these could be encouraged heavily if PAI-1 50U/50μL was injected daily. In addition, PAI-1 had no effect on that of monocularly covered eyes. Histological measurement had shown that the posterior sclera of normal chicks consisted of two parts： inner cartilaginous parts and outer fibril parts. In pure FDM group the former would become thicker and while the later become thinner and then become fibrillated magnificently, which would also be always observed in groups treated by PAI-1. What was seen in groups treated by uPA was similar to that in normal norcovered group. Immunohistochemistry had shown that abundant positive messenger dying for TGF-β1 could be found in the posterior sclera, especially adjacent to uvies membrane in normal non-covered eyes, and while in this region which would be reduced much more in pure FDM group. After injecting uPA both in normal non-coverd groups and in covered ones the positive messenger for TGF-β1 increased magnificently, and while administration of PAI-1 would be on the contrary. The content of TGF-beta messenger RNA （mRNA） was reduced heavily in FDM eyes compared with non-deprived eyes （P〈0.01）, which would disappear almost completely after daily administration of uPA, while PAI-1 could only have an inhibitor effect on that in non-covered eyes. CONCLUSION： Through inducing inner TGF-β1 expression on the posterior sclera, uPA could almost inhibit the development of chick FDM, suggesting that change in inner TGF-β1 expression on posterior sclera is associated closely with the develooment of chick FDM.