【作者】 徐韶琳；

【导师】 张晓光；

【作者基本信息】 吉林大学 ， 眼科学， 2007， 博士

【摘要】 慢性青光眼是一种伴随着视野损害和眼压升高的视神经病变。它起病缓慢，患者无明显自觉症状，目前发病机制仍不清楚。由于众所周知的原因，我们不能对活体的慢性青光眼进行更进一步的细致研究。鼠眼的房水引流途径及青光眼的眼底改变与人眼有许多相似之处。因此，我们采用烧灼大鼠表层巩膜静脉法成功建立慢性青光眼模型，进而研究MMPs及其抑制剂在大鼠慢性青光眼模型筛板组织中的表达。本课题以成功建立大鼠慢性青光眼模型为前提，通过免疫组化染色，Western Blotting及RT-PCR方法，分别在组织水平，蛋白质水平和分子水平上显示了：MMP-2、MMP-9、TIMP-1、TIMP-2在筛板组织中表达的情况。并应用免疫组化和TUNEL原位凋亡检测方法证明了孕酮对视网膜神经节细胞具有保护作用。本实验为揭示青光眼发病机制和探讨青光眼视神经的保护提供了依据。通过本课题研究，我们相信未来可以研发一种药物，经玻璃体腔或者球后途径，来增加筛板组织中MMPs的表达或抑制TIMPs的表达，进而降解筛板ECM，达到降低眼压保护视神经的作用。未来我们还可以将人工合成的雌激素应用于临床，进而为青光眼治疗提供更多的治疗手段。更多还原

【Abstract】 Chronic glaucoma including primary angle-closure glaucoma and primary open angle glaucoma is a common and frequently occur -ing disease in ophthalmology. It onsets slowly and the patients usually have no obvious subjective symptom. The pathogenesy is not clear now. It causes introcession of optic nerve head, optic atrophy and defect of field vision with optic neuropathy accompanied by intraocular hypertension. Dilatory treatment will lead to loss of sight at last. As is fairly known to us all, full intravital research on chronic glaucoma is impractical.Experimental animals are the foundation and important pedestal condition of medical science and life science. It is necessary to have suitable animal model of glaucoma to research pathogenesy, early diagnosis and treatment. Though the primate’s model is still most similar to human glaucoma model, the application is restricted by the cost, while the application of rat glaucoma model is extensive because of it’s low cost. Furthermore, anatomic structure of rat is similar to man with its blood supply of anterior segment, aqueous drainage system, episcleral venous plexus, trabecular meshwork, Schlemm’s tube. First of all ,before establishing the rat glaucoma model, we measured the normal intraocular tension of Wistar rat in conscious condition and the fluctuating rule in 24 hours according to the clinical diagnosis of glaucoma. This will provide reference to foundation intraocular tension of rat glaucoma model. The chronic ocular hypertension model is made by cauterizating three episcleral veins, which is extensively and easily put in use. This maturate method is able to provide experimental model at tissue and molecular level.Though the pathogenesy of chronic glaucoma is not clear now, the characteristic changes take place in optic never axon and connective tissue of lamina cribrosa. The changes include concentration and stagnation of axoplasma flow,axon loss of retinal ganglion cells, lamina cribrosa tumbling and remodeling of ECM in lamina cribrosa. The essential components of lamina cribrosa are elastin I, III,V,VI type collagen. The collagen wrap basement membrane is components of IV type collagen,laminin and heparan sulfate proteoglycan. Changes of normal ECM induced by ocular hypertension showed the increase of I, III, IV, VI type collagen quantitatively and the breakage, decreaseand granulation of elastin. And the tolerance of lamina cribrosa sclera is changed consequentially . In normal condition, ECM keep the dynamic balance with generating and degradating .In abnormal condition,the tissue functions are affected by stacking of ECM excessively, which induced by increase of ECM and/or decrease of degradation.Matrix metalloproteinases is a constructional and functionalgroup of zinc-dependent endopeptidases isogenesis. They can degrade lots of components of ECM. It is believed that MMPs are the most important kind of degrading ECM. Recent research indicate that the distribution of MMPs is abroad in human eyes. There are expressions of MMPs and it’s inhibitor TIMP in uveoscleral outflow , trabecular meshwork, aqueous humor, vascular smooth muscle ,optic nerve. MMP-2 and MMP-9 are gelatinases. MMP-2 is secreted by connective cells mostly, and MMP-9 is secreted by neutrocyte and macrophage mostly.They degrade gelatin I,II,III, VI type collagen and basement membrane components .MMPs and TIMPs are important inhibiting factors. TIMP-1 can inhibit activities of most MMP, and the activaties of itself can be upregulate by many cell factors.The research of Elaine. Johnson etc. indicate that there is com-Pareability between rat and primates in composition of lamina cribrosa and in reaction of ECM under ocular hypertension. This supported the relationship of glaucoma and rat model which is well used in the research on ECM, MMPs and TIMPs.The second part is to examine expression of MMP-2, MMP-9, TIMP-1, TIMP-2 in lamina cribrosa of the rat model by immuno-histology , Western blot and RT-PCR technique respectively .We confirmed that of the expressions of MMP-2,MMP-9,TIMP-1,TIMP-2 in lamina cribrosa both in normal eyes and sham eyes were little. But expressions of MMP-2,MMP-9,TIMP-1,TIMP-2 in rat lamina cribrosa in model of chronic ocular hypertension were significantly increased. Our experiment was completed in organization, protein and molecular level respectively. The reason may be the sediment of ECM in lamina cribrosa caused by ocular hypertensina of glaucoma, which induced the expressions of MMPs and TIMPs to increase. We can speculate that there is some protective mechanism which upregu-late the expression of MMPs. But the details of the mechanism is still not clear, so the futher research is necessary.Glaucoma provoke the vision loss gradually, and finally it induces the loss of sight. The main reason is the apoptosis of retinalgan-glion cells （RGCs）. Fairly significant, the multitude hypothesizes are the toxic effect of glutamic acid, interruption of neurotrophic factors, abnormality of blood supply, activation of reactive gliocyte and toxic effect of nitrogen monoxidum.The pathoalter induce the apoptosis of RGCs, and it initiate glaucoma finally.Studies in recent years indicate that estrogen protect neurocyte and prevent nerves from degenerating as neuroregulator and neuroprotective agent. As a kind of estrogen and neurosteroid at the same time, the neuroprotective effect of progesterone in peripheral nerveous system have been confirmed generally. We have paid more attention to the effect of PROG in antioxygen ,anti-ischemic and neurotrophy .We made PROG as the neuroprotective agent in the third part of the experiment ,and studied neuroprotective effect of PROG in rat RGCs in the chronic glaucoma model. After we setting up the model of chronic glaucoma, rats had intraperitoneal infections of different level of PROG. 12 weeks later, the rats were executed and the eyeballs were taken out. The neuroprotective of PROG to RGCs would be detected by immunohistological technique and TdT-dUDP terminal nick end-labeling （TUNEL） technique. The result displayed that PROG could provide neuroprotection for RGCs in the rat chronic glaucoma model and the neuroprotection may have the positive correlation with the level of PROG.At present ,analog of Prostaglandin F_2α is a kind of new drug exploited resent years. The main mechanism is that it promote ciliaris muscle to secret MMPs , increase the coefficient of aqueous outflow facility which reduce ocular hypertension. Our experiment confirmed that the expressions of MMP-2,MMP-9,TIMP-1,TIMP-2 in rat lamina cribrosa in model of chronic ocular hypertension were significantly increased. It is believed that in the future, we are able to research and exploit a neuroprotective drug which increase the expression of MMPs but reduce the expression of TIMPs and degrade extracellular matrix（ECM） in lamina cribrosa via the channel of vitreous body or posterior eyeball.Many researches indicate that neuroprotective of estrogen is definite. But we found that HRT had increase the incidence of cerebral apoplexy clinically. SO the clinical application of HRT had been confined. At present, some artificial estrogens get rid of the malpractices of natural estrogen and have the same neuroprotection. Therefore artificial estrogen may have enormous potential in curing vision functional disorder and neuroprotective effect.更多还原