【作者】 王莹；

【导师】 赵堪兴；

【作者基本信息】 天津医科大学 ， 眼科学（专业学位）， 2012， 博士

【摘要】 研究目的病理性近视(Pathologic myopia, PM)是成年人重要的致盲性眼病之一,目前尚无良好治疗对策。近视发病机制的研究提示：巩膜是各种致病因素作用的主要靶器官,巩膜病理性的薄弱是PM作用的结果,也是导致其它相关病变的诱因,而针对巩膜生物力学强度变化的干预有可能是阻断PM进展的一条可行途径。本研究通过建立豚鼠形觉剥夺性近视(form deprivation myopia, FDM)模型,应用甘油醛促进巩膜交联,探讨甘油醛对巩膜生物力学特征的影响及其对实验性近视的抑制作用,从控制巩膜重塑并增加其强度的角度,为防治PM提供新思路。研究内容和方法1.甘油醛对离体巩膜条带的交联作用：选用15只三周龄三色豚鼠,按随机数字表法分成3组,每组5只。均取右眼为实验眼。矢状位取2mm×6mm的巩膜条带,给予0.005mol/L甘油醛组,0.05mol/L甘油醛组和空白对照液室温下浸泡4天。应用微材料力学性能测试系统,对各组巩膜条带的极限应力(σmax (MPa)、极限应变εmax (%)、6%弹性模量E(MPs)进行统计分析。2.豚鼠FDM模型的建立：用6号乳白色半透明乳胶气球,依照豚鼠头部形状剪制成头套,暴露出动物的左眼,双耳及口鼻。制作动物右眼形觉剥夺模型。室温下饲养,正常昼夜节律。分别于实验开始前、形觉剥夺7、14、21天时测量眼轴长度、玻璃体腔长度及屈光度,进行统计学分析。3.甘油醛后巩膜交联：选用35只三周龄三色豚鼠,将动物按随机数字表法分成5组,每组7只,右眼为实验眼,左眼为对照眼。A组：右眼遮盖7天；B组：右眼遮盖21天,0.9%生理盐水第1、8、15天Tenon’s囊下球后注射；C组：右眼遮盖21天,0.05mol/L甘油醛第1、8、15天Tenon’s囊下球后注射；D组：右眼遮盖21天,0.5mol/L甘油醛第1、8、15天Tenon’s囊下球后注射；E组：正常对照组。并于实验开始前、形觉剥夺7、14、21天时测量眼轴长度、玻璃体腔长度及屈光度。在实验结束时,应用微材料力学性能测试系统测量5个组巩膜的极限应力σmax (MPa)、极限应变εmax (%)、6%弹性模量E(MPa),并与对侧眼比较,进行统计学分析。绘制5个组的应力-应变曲线,比较甘油醛交联对豚鼠巩膜生物力学强度的影响。4.后巩膜交联术对豚鼠FDM眼超微结构影响的观察：选用18只三周龄三色豚鼠,随机抽取其中的15只豚鼠,按随机数字表法分成5组。右眼为实验眼,左眼为对照眼。实验方法同3。实验结束后处死豚鼠,在视神经颞侧连续切取5片组织,厚度0.5μm,2片行苏木精-伊红染色,3片行免疫组化染色,检测后极部巩膜、脉络膜、视网膜MMP-2表达,光学显微镜下观察并照相。余下的3只豚鼠给予0.5mol/L的甘油醛Tenon’s囊下球后注射,分别在第3、7、14天处死。取注射相邻部位的角膜缘、眼外肌和视神经筛板做切片,行苏木精-伊红染色。判断甘油醛对视网膜、视神经是否有毒性。结果1.离体巩膜实验：不同浓度甘油醛组与对照组实验眼巩膜的应力-应变曲线有明显的梯度变化。空白对照组巩膜的极限应力σmaax、极限应变smax、6%弹性模量E分别为(7.198±0.991)MPa、(21.480±0.853)%和(22.808±2.159)MPa。甘油醛交联组中,6%弹性模量E0.005mol/L甘油醛组、0.05mo/L甘油醛组分别为(25.903±0.892) MPa (P=0.019)和(36.156±2.026) MPa (P=0.000),较空白对照组分别增加了13.57%和58.52%。0.005mol/L甘油醛组、0.05mo/L甘油醛极限应力σmax分别为(10.386±1.023)MPa(P=0.0012)和(14.851±1.602)MPa (P=0.000),较空白对照组分别增加了44.30%和106.32%；0.005mol/L甘油醛组、0.05mo/L甘油醛组极限应变smax分别为(19.320±±0.672)%(P=0.002)和(14.140±±1.001)%(P=0.000),较空白对照组分别降低10.06%和34.17%。2.形觉剥夺后,实验眼均出现了玻璃体腔长度和眼轴长度的增加以及相对近视度数的增加,在A组、B组和C组中,玻璃体腔长度、眼轴长度和屈光度与对侧眼相比,差异有统计学意义(P玻璃体腔=0.018,0.002,0.001；P眼轴=0.019,0.002,0.000；P屈光=0.000,0.005,0.001)。D组、E组玻璃体腔长度、眼轴长度与对侧眼相比,差异无统计学意义(P玻璃体腔=0.607,0.539；P眼轴=0.607,0.539)D组实验眼与对侧眼屈光度相比差异有统计学意义(P屈光=0.020)。3.在实验第21天结束时,B、C、D、E组实验眼的屈光度变化值分别为(8.800±0.616)D,(7.236±2.198)D,(6.271±1.112)D和(0.934±0.158)D,总体差异有统计学意义(F=61.249,P=0.000),B、C、D组的屈光度变化值较正常对照组大,差异有统计学意义(PB=0.000,PC=0.000,PD=0.000)；B、C、D组的眼轴变化值较正常对照组大,但差异无统计学意义(PB=0.430,PC=0.840,PD=0.386)4.在实验第21天结束时,B组和E组对侧眼比较,B组玻璃体腔长度的增加值为(0.198±0.038)mm,E组为(0.139±0.026)mm,差异有统计学意义(t=-3.689,P=0.002)；B组眼轴长度的增加值为(0.481±0.062)mm,E组为(0.438±0.068)mm,差异无统计学意义(t=-1.295,P=0.216)5.B组实验眼眼轴长度与屈光呈高度负相关(r=-0.832,P=0.000)；B组实验眼玻璃体腔长度与屈光度呈高度负相关(r=-0.804,P=0.000)；E组右眼的眼轴长度和屈光度呈中度负相关(r=-0.604,P=0.000)；E组双眼的眼轴长度呈高度相关(r=0.940,P=0.000)。6.试验结束时,B组实验眼的极限应力和6%弹性模量分别为(7.988±3.677)MPa(P=0.002)和(19.938±4.871)MPa(P=0.001),较对侧眼分别降低了34.21%和34.38%,极限应变实验眼为(28.6±3.6)%(P=0.034),较对侧眼增加了19.17%。甘油醛交联后,C组实验眼的极限应力和6%弹性模量分别为(9.244±0.806)MPa(P=0.001)和(26.180±4.388)MPa(P=0.031),较对侧眼分别降低了23.13%和13.34%,极限应变实验眼为(26.2±1.0)%(P=0.016),较对侧眼增加了12.93%；D组实验眼的极限应力为(12.476-2.507)MPa(P=0.580),较对侧眼降低了5.50%,6%弹性模量为(30.446±3.410)MPa(P=0.314),较对侧眼增加了6.53%,极限应变为(23.8±1.8)%(P=0.253),较对侧眼降低了4.42%。在D组(0.5mol/L甘油醛)攻膜交联21天后与B组的实验眼相比,巩膜的极限应力增加了35.85%,6%弹性模量增加了52.70%,极限应变降低了16.78%。不同浓度甘油醛交联与对照组实验眼的应力-应变曲线有明显的梯度变化,D组的应力-应变曲线接近正常对照组,高于其它3组。7.组织学检查：随着豚鼠FDM的出现,表现为眼外组织疏松,巩膜厚度明显降低,纤维排列整齐,脉络膜厚度降低,可见血管腔,但未见大的窦腔,视网膜层次清晰。MMP-2在眼球表层结缔组织、巩膜表层结缔组织、浅层巩膜、脉络膜实质层以及视网膜外丛状层阳性表达比正常对照眼明显。甘油醛交联后出现了眼球表面结缔组织结构变得相对致密,排列有序,与巩膜连接紧密。巩膜组织的厚度变薄,细胞密度增加。脉络膜管腔扩大,厚度变化不明显。视网膜光感受器细胞核清晰,排列整齐,无炎性细胞浸润。MMP-2在除眼表结缔组织外的巩膜表面结缔组织,浅层巩膜、脉络膜实质层以及视网膜外丛状层的阳性表达明显降低。结论：1.甘油醛是一种安全有效的交联剂,能够显著增强巩膜的生物力学强度。2.头套法形觉剥夺成功诱导出以豚鼠为对象的动物近视模型,而且这种近视是以眼轴延长特别是玻璃体腔长度增加为主的轴性近视。3.随着眼轴的延长及近视度数的加深,巩膜的生物力学强度明显降低。4.本实验进一步证明了MMP-2与近视形成的相关性,而且发现甘油醛能够降低MMP-2的阳性表达。5.采用甘油醛交联的方法能够有效控制FDM动物模型眼近视的发展。为防治PM提供新思路。更多还原

【Abstract】 [Objective]Pathologic myopia (PM) is one of the most serious eye disease to blindness in the clinic, especially for adults. There is no clinical successful therapeutic intervention that benefit at present. Studies on pathogenesis had shown that various agents had taken sclera as the essential target structure, and the weakness of sclera might be either the result of PM, or of the risk factor that could induce related lesions. Therefore, enhancement of sclera biomechanical strength could be as a referenced method to alleviate of the development of PM.The purpose of this study is to observe the effects of glyceraldehyde cross-linking on sclera biomechanical strength and experimental myopia, based on the model of the guinea pig form deprivation myopia (FDM). New therapeutic strategy for PM was explored from the view of scleral remodeling control.[Materials and Methods]1In vitro experiment:15three-color guinea pigs aged three weeks were randomly divided into3groups. Each group had5guinea pigs. The right eyes were treated and the left eyes were used as untreated controls.2mm x6mm sclera strip was taken after the eyeball extraction and treated respectively with glyceraldehyde at concentration of0.005mol/L,0.05mol/L, and foundational solution without glyceraldhyde at room temperature. After4-day cross-linking, biomechanical stress-strain measurements of all scleral strips were performed using a microcomputer-controlled biomaterial testing device. The parameters ultimate stress (σmax)(MPa) and ultimate strain (εmax)(%) and6%elastic modulus (MPa) were used for analysis.2In the MDF group, latex balloons were modified into facemasks, which only covered the right eye of the animals, leaving the left eye, nose, mouth and ears exposed. Guinea pigs breeding at room temperature, normal circadian rhythms Ocular axial length, vitreous cavity length and refractive error were measured before form deprivation and at mask day7,14,21.3Posterior scleral cross-linking:35three weeks aged guinea pigs were randomly divided into5groups:A, B, C, D, E. Each group had7guinea pigs. The right eye was set as the experimental eye, the left eye was used as control. Group A:7days mask; Group B:21days mask, plus physiological saline retrobulbar injection at mask day1,8,15; Group C:21days mask, plus0.05mol/L glyceraldehyde retrobulbar injection at mask day1,8,15; Group D:21days mask, plus0.5mol/L glyceraldehyde retrobulbar injection at mask day1,8,15; Group E:normal control group. Ocular axial length, vitreous cavity length and refractive error were measured before form deprivation and at mask day7,14,21.2mm x6mm sclera strip was taken after the eyeball extraction at the end of experiment, biomechanical stress-strain measurements of all scleral strips were performed using a microcomputer-controlled biomaterial testing device. The parameters ultimate stress (amax)(MPa) and ultimate strain (εmax)(%) and6%elastic modulus (MPa) were used for analysis.4Ultrastructure examination:15three weeks aged guinea pigs were randomly divided into5groups. The right eye was set as the experimental eye, the left eye was used as control. Administration of posterior scleral cross-linking was performed referring to protocols described in step3. Animals were humanly killed at the end of the experiment. Ocular ultrastructure structures examination of sclera, choroid, retina was performed by hematoxylin-eosin staining. The expression of MMP-2was detected by immunohistochemical staining. The effects of glyceraldehyde on adjacent tissues, including limbus, the extraocular muscles and optic nerve were also detected.[Results]1. The stress-strain curves showed the prominent increase of biomechanical stiffness in vitro. After the cross-linking treatment, the ultimate stress, the ultimate strain,6%elastic modulus were (7.198±0.991)MPa,(21.480±0.853)%and (22.808±2.159) MPa in the controls.6%elastic modulus of0.005mol/L. glyceraldehyde group and0.05mol/L glyceraldehyde group were (25.903±0.892)MPa(P=0.019)and(36.156±2.026)MPa(P=0.000), increased13.57%and58.52%versus the control group respectively. The ultimate stress of0.005mol/L glyceraldehyde group and0.05mol/L glyceraldehyde group were (10.386±1.023) MPa (P=0.0012) and (14.851±1.602) MPa (P=0.000) increased44.30%and106.32%respectively. The ultimate strain of groups0.005mol/L, glyceraldehyde group and0.05mol/L glyceraldehyde group were (19.320±0.672)%(P=0.002) and (14.140±1.001)%(P=0.000), decreased10.06%and34.17%respectively.2. Form deprivation, at the end of the experiment, the experimental eyes appeared the increase of the the vitreous cavity length, the axial length and myopia. In group A, group B and group C. the length of the vitreous cavity, the axial length and refractive error, compared with the fellow eye the difference was statistically significant statistically (Pvitreous cavity=0.018.0.002.0.001; Paxial=0.019.0.002.0.000; Prefraction=0.000,0.005,0.001). In group D and group E, the length of the vitreous chamber, axial length compared with the fellow eye, the difference was not statistically significant (P vitreous cavity=0.607,0.539; Paxial=0.607.0.539). The experimental eye and the fellow eye of group D, the difference of diopter was statistically significant (Prefraction=0.020), but diopter of group E was no significant difference.(Prefraction=0.580).3. At the end of the experiment, diopter change in value of experimental eye of group B, C, D, E was significantly different, the overall difference was statistically significant (F=61.249, P=0.000). Diopter change value of group B,C,D versus the normal control group, the difference was statistically significant (PB=0.000. Pc=0.000, PD=0.000):but the difference of axial length changes in the value was no statistical significance (PB=0.430, Pc=0.840. PD=0.386).4. At the end of the experiment, B and E groups contralateral eye, the added value of group B of the vitreous cavity length compared with group E, the difference was statistically significant (t=-3.689, P=0.002); the difference of the increase in axial length was not statistically significant (t=-1.295, P=0.216). 5. Axial length and refractive error of experimental eye of group B was highly negative correlated (r=-0.832, P=0.000); vitreous cavity length and refractive error of experimental eye of group B was highly negative correlated (r=-0.804, P=0.000);the axial length and refraction of right eye of group E was a moderate negative correlation (r=-0.604, P=0.000); axial length of group E eyes was highly correlated (r=0.940, P=0.000).6. At the end of the experiment, the ultimate stress and6%elastic modulus of group B experimental eye was (7.988±3.677) MPa (P=0.002) and (19.938±4.871) MPa(P=0.001), decreased10.06%and34.17%versus the fellow eye respectively, the Ultimate strain was (28.6±3.6)%(P=0.034) increased19.17%respectively. After the cross-linking treatment, the ultimate stress and6%elastic modulus of group C experimental eye was (9.244±0.806)MPa(P=0.001)and(26.180±4.388) MPa (P=0.031). decreased23.13%and13.34%respectively. the ultimate strain was (26.2±1.0)%(P=0.016).increased12.93%respectively. The ultimate stress of group D experimental eye was (12.476±2.507) MPa (P=0.580).decreased5.50%,6%elastic modulus was (30.446±3.410) MPa (P=0.314) increased6.53%, ultimate strain was (23.8±1.8)%(P=0.253). decreased4.42%respectively. The ultimate stress of group D experimental eye increased35.85%versus group B,6%elastic modulus increased52.70%, ultimate strain decreased16.78%. The stress-strain curves showed the prominent increase of biomechanical stiffness.7. Ultrastructure examination:As the development of guinea pig FDM, Ultrastructure examination showed that, decreased scleral thickness with fibers lined up in order, decreased choroidal thickness with vascular lumen, lack of sinus cavity. The retinal structure was normal. Expressions of MMP-2were mainly in the episcleral tissue, the stroma of choroid, and the outer plexiform layer. After glyceraldehyde cross-linking, the loose episcleral tissue became dense and structured, and the adhesion with sclera became tight. Scleral thickness decreased while the dense of sclera cells increased. Lumens and cavities in the choroid became obvious, and no significant thickness change was noticed, The retinal structure was morphologically normal with distinct structural layers and regular arrangement of retinal photoreceptor cells, without inflammatory cells Infiltration. Expressions of MMP-2were mainly in the episcleral tissue. MMP-2in the stroma of choroid, and the outer plexiform layer.[Conclusion]1. Glyceraldehyde is a safe and effective cross-linking agent that could significantly enhance the sclera biomechanical strength.2. The monocularly-deprived facemask could successfully induce guinea pig for animal myopia model, which is axial extension myopia, especially the vitreous cavity length.3. With increasing of the axial length and progressing of myopia, the scleral biomechanical strength is significantly reduced.4. The correlation between MMP-2and progression of myopia model is meaningful, and glyceraldehyde could reduce the positive expression of MMP-2.5. Glyceraldehyde cross-linking method could effectively control the development of pathologic myopia in animal model. New therapeutic strategy for PM was explored from the view of scleral remodeling control.更多还原