【作者】 陈悦；

【导师】 张金嵩；

【作者基本信息】 郑州大学 ， 眼科学， 2007， 博士

【摘要】 近视是眼科的常见病、多发病。高度近视又称病理性近视或变性近视。病理性近视病理改变主要有后巩膜葡萄肿、视网膜和脉络膜萎缩变性、黄斑变性、脉络膜新生血管、孔源性视网膜脱离等严重并发症而致失明。形觉剥夺性近视（form deprivation myopia，FDM）模型的建立，为探索近视的发病机制提供了新途径。形觉剥夺使存在于局部视网膜的多种神经递质与生长因子水平发生改变，它们通过互相平行或彼此交叉的途径发挥作用。经视网膜-脉络膜-巩膜复杂而精细的调控机制，作用于巩膜，引起巩膜生物化学和超微结构的改变，巩膜主动重塑，眼轴延长，引起轴性近视。随着眼轴长度进行性增长和眼底病变的发展，视网膜光感受器细胞减少，视网膜细胞的凋亡是退行性变性疾病的重要病理特征。现今对其光感受器细胞死亡的机制及相应的药物治疗尚缺乏深入细致的研究。神经生长因子（never growth factor，NGF）是神经营养素家族成员之一，具有调控神经元和非神经元的分化、增殖和维持其功能的双重作用。在视网膜感光细胞、视网膜神经节细胞、双极细胞三级神经元均有表达。Nastri将抗鼠NGF免疫血清通过局部点眼的方式阻止了鸡近视的发展，提出了NGF可能参与近视形成的推测。已知NGF的受体p75NTR与神经细胞凋亡密切相关，外源性NGF抑制实验性视网膜脱离的视网膜细胞凋亡。本实验在建立豚鼠形觉剥夺性近视模型的基础上应用免疫组织化学和RT-PCR的方法从蛋白质和RNA水平研究NGF与受体TrkA、p75NTR在FDM中的动态表达，首先探讨NGF与TrkA在FDM中的发生、发展的关系，然后研究了FDM发展过程中视网膜光感受器细胞是否也存在细胞凋亡现象及凋亡基因p75NTR、Caspase-3蛋白和mRNA表达的变化，并观察了NGF对视网膜细胞的保护作用，探索治疗高度近视的新途径。最后研究了NGF对体外培养的人巩膜成纤维细胞的影响。第一部分神经生长因子及其受体在豚鼠形觉剥夺性近视的表达方法1．动物模型的建立：选用出生7天花色豚鼠80只。随机分为4组，每组20只。A组（遮盖2周），B组（遮盖4周），C组（遮盖8周），D组（遮盖7周去遮盖1周）。每组10只用于HE染色及免疫组织化学检测，另10只用于RT-PCR检测。右眼以半透明眼罩遮盖作为遮盖眼，左眼不做任何处理为对照眼。2．屈光度及眼轴长度测量：分别于遮盖前、遮盖后2周、4周、8周及遮盖7周去遮盖1周，检影镜检测豚鼠双眼屈光状态，A型超声仪测定双眼轴长度。3．HE染色与形态学观察：光学显微镜下观察遮盖前后视网膜形态学变化。4．免疫组织化学染色检测：NGF蛋白、TrkA蛋白表达。5．逆转录聚合酶链反应（RT-PCR）检测：视网膜NGF mRNA、TrkA mRNA的表达。6．统计学处理：应用SPSS12.0统计软件进行统计学处理，数据以均数±标准差（（?）±s）表示，双眼比较采用配对t检验，组间比较采用单因素方差分析，取α=0.05作为检验标准。结果1．遮盖眼与对照眼屈光度及眼轴比较：遮盖眼由实验前远视眼演变为近视眼，并随遮盖时间延长，近视度数加深，眼轴延长。遮盖8周，遮盖眼屈光度（-4.38±1.11）D与对照眼（3.75±0.71）D相比，眼轴长（8.18±0.16）mm与对照眼（7.26±0.20）mm相比屈光度前后变化为8.13D，眼轴前后变化为0.92mm，差异有统计学意义（P＜0.01）。遮盖7周去遮盖1周屈光度前后变化为5.91D，眼轴前后变化为0.52mm。去遮盖眼屈光恢复27％，眼轴长恢复43％。2．视网膜、巩膜形态学变化：遮盖眼视网膜较对照眼变薄，后极部巩膜变薄、稀疏。3．免疫组织化学染色：NGF、TrkA阳性细胞主要位于视网膜神经节细胞层、内核层，表现为细胞浆呈棕褐色。经图象系统分析处理，遮盖8周和去遮盖1周眼视网膜NGF蛋白、TrkA蛋白表达减少，与对照眼相比差异有统计学意义（P＜0.01）。4．RT-PCR：遮盖眼视网膜NGF mRNA、TrkA mRNA表达水平较对照眼降低，与对照眼相比差异有统计学意义（P＜0.05）。第二部分形觉剥夺性近视豚鼠视网膜细胞凋亡和神经生长因子的保护作用研究一豚鼠形觉剥夺性近视视网膜细胞凋亡和NGF受体p75NTR、Caspase-3的表达方法1．动物模型的建立：60只出生7天花色豚鼠随机分为3组：A组（遮盖4周组）、B组（遮盖8周组）、C组（遮盖12周组），每组20只。右眼以半透明眼罩遮盖作为遮盖眼，左眼不做任何处理为对照眼。余同第一部分。2．屈光度及眼轴长度测量：同第一部分3．末端脱氧核苷酸转移酶介导d-UTP缺口末端标记（Terminal deoxynucleotidyl transferase mediated x-dUTP nick end labeling，TUNEL）染色技术：检测视网膜细胞凋亡。4．免疫组织化学染色：检测p75NTR蛋白、Caspase-3蛋白表达。5．逆转录聚合酶链反应（RT-PCR）：检测视网膜p75NTR mRNA的表达。6．统计学处理：同第一部分。结果：1．各实验组遮盖眼分别与对照眼相比屈光度加深、眼轴增长，C组遮盖12周诱导出-11.47D近视，眼轴增长1.06mm，且其脉络膜、视网膜变薄，光感受器外节变长，视网膜色素上皮（Retinal Pigment Epithelium，RPE）细胞排列紊乱。2．TUNEL染色仅在C组遮盖眼视网膜内、外核层检测到凋亡细胞，对照眼、A组、B组遮盖眼未见到凋亡细胞。3．C组p75NTR蛋白、Caspase-3蛋白表达阳性染色呈棕褐色，主要位于神经节细胞层、内核层、外核层。与对照眼、A组、B组遮盖眼比较差异有统计学意义（P＜0.05）。4．对照眼及A组、B组遮盖眼有极弱p75NTR mRNA表达，C组遮盖眼后极部视网膜p75NTR mRNA表达增强，与对照眼及A组、B组遮盖眼比较差异有统计学意义（P＜0.05）。研究二神经生长因子对豚鼠形觉剥夺性近视细胞凋亡及凋亡相关基因表达的影响方法1．120只出生7天花色豚鼠随机分为6组：A组（正常对照组）、B组（遮盖12周组）、C组（遮盖12周+生理盐水组）、D组（遮盖12周+NGF5μg组）、E组（遮盖12周+NGF10μg组）、F组（遮盖12周+NGF20μg）组，每组20只。除A组外，各组均右眼以半透明眼罩遮盖作为遮盖眼，左眼不做任何处理为对照眼。按预定时间遮盖眼玻璃体腔注射NGF或生理盐水后检影验光，测量眼轴长度。2．流式细胞术检测各组视网膜细胞凋亡情况。3．免疫组织化学染色法检测TrkA蛋白、p75NTR蛋白、Caspase-3蛋白表达。4．逆转录聚合酶链反应检测视网膜TrkA mRNA、p75NTR mRNA的表达。5．统计学处理同第一部分，凋亡率应用多个样本比较秩和检验。结果：1．B组、C组、D组、E组、F组遮盖眼分别与对照眼、A组正常眼比较，其屈光度加深、眼轴增长。2．B组、C组视网膜细胞凋亡率为（4.45±0.48）％和（4.56±0.32）％，高于正常对照A组（0.31±0.22）％（P＜0.05）。D组、E组及F组NGF玻璃体腔注射后，遮盖眼视网膜细胞凋亡率均降低：F组（1.05±0.26）％，E组（1.94±0.42）％，D组（3.22±0.34）％，与A组及B组、C组差异有统计学意义（P＜0.05）。3．B组、C组遮盖眼视网膜p75NTR蛋白、Caspase-3蛋白阳性表达高于A组（P＜0.05）。D组、E组及F组玻璃体腔注射NGF后，p75NTR蛋白、Caspase-3蛋白的阳性表达降低，TrkA蛋白升高，但与A组及B组、C组比较差异有统计学意义（P＜0.05）。4．D组、E组及F组NGF玻璃体腔注射后p75NTR mRNA表达降低，TrkA mRNA表达升高，与A组及B组、C组比较差异有统计学意义（P＜0.05）。第三部分神经生长因子对体外培养的人巩膜成纤维细胞的影响方法1．选取12周～18周人胚胎眼球，体外培养HSF，进行原代及传代HSF的形态学观察和波形蛋白免疫组织化学检测以鉴定是否为巩膜成纤维细胞。2．取第3～6代的细胞，MTT法观察NGF对体外培养HSF增殖的质量浓度、时间效应关系。3．流式细胞仪测定NGF对HSF细胞周期的影响。4．氯胺T法检测NGF对HSF胶原合成的影响。5．统计学处理同第一部分。结果1．原代、传代培养人胚胎巩膜成纤维细胞生长情况良好，波形蛋白免疫组织化学染色阳性。2．MTT法检测结果：NGF在25μg／L～200μg／L浓度范围内促进HSF的增殖且呈剂量效应关系，以100μg／L浓度时促进增殖作用最显著（P＜0.01）。流式细胞检测发现NGF100μg／L作用下G₁期细胞比例下降，S期细胞比例增加，反映细胞增殖状况的细胞增殖指数提高。3．NGF在第3天开始与对照组相比对巩膜成纤维细胞有显著的促增殖作用（P＜0.05）。4．NGF在25μg／L～200μg／L与对照组相比能有效刺激巩膜成纤维细胞胶原蛋白合成，并呈剂量效应关系。当浓度为200μg／L时，作用达到饱和。结论1．成功建立了豚鼠FDM的模型并在形态学上和生物特性方面进行了验证。2．在时空分布上从蛋白表达和RNA水平方面研究证实了NGF和功能性受体TrkA参与FDM的形成与发展。遮盖眼视网膜NGF蛋白、TrkA蛋白明显减少；NGFmRNA、TrkA mRNA表达水平明显降低。3．形觉剥夺可导致高度近视，遮盖眼视网膜内、外核层细胞出现凋亡细胞，p75NTR蛋白和Caspase-3蛋白明显增加，p75NTR mRNA表达水平明显升高。4．外源性NGF可能通过下调p75NTR与Caspase-3表达，上调TrkA表达而有效抑制形觉剥夺性高度近视视网膜细胞的凋亡。5．NGF可以促进HSF增殖并呈时间剂量效应依赖关系。其促细胞增殖作用可能与NGF对细胞周期的调节有关，在一定浓度范围内能有效刺激HSF胶原蛋白合成。更多还原

【Abstract】 Myopia is commonly and frequently encountered disease in ophthalmology,especially high myopia, which is also called pathological myopia or degenerationmyopia. High myopia is often associated with excessive and progressive elongation ofthe eye globe, resulting in varying degrees of visual deterioration. It can give rise tomany complications leading to blindness, such as degenerative retinopathy, posteriorscleral staphyloma, choroidal neovascularization, macular degeneration andrhegmatogenous retinal detachment, etc. The foundation of form deprivationmyopia（FDM）model is helpful for us to further understand how high myopia occursand develops. Form deprivation modulates the development of local sclera by changing the level of various neurotransmiter and growth factors in the retina. Theseneurotransmiter and growth factors work by means of cooperate parallel and across,and lead to the ultrastructural and biomechanical alteration of sclera through a seriesof signal transduction. Sclera was remodeled and eye axial was lengthened andinduced to axis myopia. With the development of the axial length of eye and funduschanges, retinal photoreceptor cells decrease continuously. Apoptosis is one of thepathways of photoreceptor cell death in retinal degenerated disorders. At present, it islack of investigating the mechanisms of retinal apoptosis and its anti-apoptosistreatment in retinal degenerative disease including high myopia.Nerve growth factor is a member of family of neurotrophins. It exerts effects bysignaling through surface membrane receptors, trkA and p75NTR in the neurotissueand unneurotissue. NGF expresses and influences the shape and function of the threekinds neuron of in the retina. Nastri had discovered that Anti-NGF serum eye dropswere able to elicit a slight contraction on ocular overgrowth in chicks raised incontinuous light and suggested the NGF was possible to take part in the developmentof FDM. It is well known that NGF receptor p75NTR involved in programmed celldeath （PCD） and exogenous NGF may inhibit the apoptosis of retinal cell inexperiment retinal detachment.In this study, form deprivation myopia model is established to investigateexpression of NGF、TrkA、p75NTR and Caspase-3 protein in retina byimmunohistochemistry, and to detect the expression of NGF mRNA、TrkA mRNAand p75NTR mRNA in retina by reverse transcription-polymerase chainreaction（RT-PCR）. We would investigate whether there are obvious retinal apoptosisin FDM in guinea pigs and observe the retinal rescue of NGF in order to provide anew additional strategy for the treatment of human high myopia. We also observedthe effect of NGF on human sclera fibroblast in vitro.Part one: Expression of nerve growth factor （NGF） and high affinityNGF receptor （TrkA） in retinal tissue of guinea pigs with form deprivation myopiaMethods1. Establishment of a form deprivation myopia model: Eighty guinea pigs（seven-day-old） were divided into 4 groups randomly, i.e, group A （2 weeks’occluded）, group B （4 weeks’ occluded）, group C （8 weeks’ occluded）, group D（7weeks’ occluded and removed for 1 week）. There were 20 guinea pigs in each group.Ten guinea pigs were used for immunohistochemistry, the other ten guinea pigs wereused for RT-PCR. All the right eyes underwent form deprivation with translucentgoggles and the left eyes were used as control.2. Measure of the refractive state and the axial length: Before occluding andafter occluding for A、B、C、D group, the refractive state and axial length weremeasured with retinoscopy and A-scan ultrasonography respectively.3. Hematoxylin-eosin staining and morphology: After the refractive state andaxial length were measured,the eyes were extracted,routine hematoxylin-eosinstaining were done, and morphological changes in form deprived guinea pigs retinaand sclera were observed under light microscope.4. The expression of NGF protein and TrkA protein in retina were detected byimmunohistochemistry.5. The expression of NGF mRNA and TrkA mRNA in retina were detected byRT-PCR.6. Statistical analysis SPSS12.0 software was used to perform t-test andanalysis of variance, less than 0.05 was considered statistical significance.Results1. Comparison of refraction state and axial length between the occluded eyesand control eyes: Monocular occluding for 8 weeks could lead to myopia, therefraction showed significant difference between the occluded eyes （-4.38±1.11）D andthe control ones(（3.75±0.71）D, the change of refractive degree was 8.13D（P＜0.01）.The longer the eyes were occluded, the axial length was longer, there was significant differences between the occluded eyes（8.18+0.16）mm and the controlones（7.26±0.20）mm, the change of axial length was 0.92mm（P＜0.01）.The longerthe eyes were occluded, the more severe the myopia was. The removal of occludingfor I week, the refraction state and axial length was recovered 27％、43％respectively.2. Morphology change in retina and sclera: hematoxylin-eosin staining showedthe posterior retina of the occluded eyes were thinner than that of the control eyes,The posterior sclera of the occluded eyes were thin too.3. Immunohistochemistry: NGF and TrkA positive cells were mainlydistributed in the inner nuclear layer and ganglion cell layer of the retina, displayingas pale brown pellet. Through the image analysis, the expression of NGF protein andTrkA protein in retina of the occluded eyes was decreased, there were significantdifference compared with that in the control eyes（P＜0.01）4. RT-PCR: The expression of NGF mRNA and TrkA mRNA in retina of theoccluded eyes decrease, there were significant difference compared with that in thecontrol eyes（P＜0.01）.Part two: Experimental study of retinal apoptosis and NGFrescue in form deprivation myopia in guinea pigsResearch one: Expression of retinal apoptosis and NGF receptionp75NTR in form deprivation myopia in guinea pigsMethods:1. Sixty guinea pigs （seven-day-old）were divided into three groups, this is, groupA （4 weeks’ occluded）, group B （8 weeks’ occluded）, group C （12weeks’ occluded）.There were 20 guinea pigs in each group. All the right eyes underwent formdeprivation with translucent goggles and the left eyes were used as control.Establishment of a form deprivation myopia model and measure of the refractive stateand the axial length: refers to part one.2. Histopathological changes in retina were observed by light microscopy. Retinal apoptotic cells were determined by TdT medicated biotin-dUTP nick-endlabeling（TUNEL） staining.3. The expression of p75NTR protein and Caspase-3 protein in retina wasdetected by immunohistochemistry.4. The expression of p75NTRmRNA in retina was detected by RT-PCR.5. Statistical analysis refers to part one.Results1. Comparison of refraction state and axial length between the occluded eyes andcontrol eyes: The refractive degrees and axial length of occluded eyes weresignificantly deeper and longer than control eyes. After occluding for 12 weeks, thechange was -11.47D in refraction degree and 1.06mm in axial length.2. The thickness of choroid and retina occluded 12 weeks became thinneraccompanying with the outer Segment of photoreceptor elongated and RPE arrangeddisorderly. Apoptotie cells were only found in retinal outer and inner nuclear layer ofthe occluded eyes in group C by TUNEL staining.3. Immunohistochemistry:p75NTR and Caspase-3 positive cells were mainlydistributed in the outer segments of photoreceptor cells of the retina,immunohistochemistry staining shows that p75NTR and Caspase-3 positiveexpression display as pale brown. Through the image analysis, the expression ofp75NTR protein and Caspase-3 protein in retina of the occluded eyes increase. Therewere significant difference compared with that in the control eyes （P＜0.01）.4. RT-PCR: The expression of p75NTR mRNA and Caspase-3 mRNA in retinaof the occluded eyes increase, there were significant difference compared with that inthe control eyes （P＜0.01）.Research two: NGF protective effect on retinal apoptosis in formdeprivation myopia in guinea pigsMethods1. One hundred twenty guinea pigs （seven-day-old）were divided into six groups, this is, group A（normal control）, group B（12 weeks’ occluded）, group C（12 weeks’occluded +saline）,group D（12 weeks’ occluded +NGF5μg）, group E（12weeks’occluded+NGF10μg）, group F（12 weeks ’occluded +NGF 20pg）. There were 20guinea pigs in each group. Except group A, all the right eyes underwent formdeprivation with translucent goggles and the left eyes were used as control. AfterNGF or saline had been injected into vitreous of occluded eyes in planning time,myopia was confirmed by retinoscopy and A-scan ultrasonography.2. Retinal apoptotic cells were determined by flow cytometry.3. The expression of TrkA proteins, p75NTR proteins, Caspase-3 proteins inretina were detected by immunohistochemical staining.4. The expression of TrkA mRNA, p75NTR mRNA in retina was detected byRT-PCR.5. Statistical analysis refers to part one. Apoptotic rate by several independentsamples ranks test.Results:1. The refractive degrees and axial length of form deprivation eyes weresignificantly deeper and longer in group B、C、D、E、F comparing with its self-controleyes and group A（P＜0.01）.2. Retinal apoptotic rate of group B（4.45±0.48）％, C（4.56±0.32）％was morethan group A（0.31±0.22）％; The rate of group D（3.22±0.34）％、E（1.94±0.42）％、F（1.05±0.26）％was less than group B、C（P＜0.01）, but more than group A.3. Retinal p75NTR proteins and Caspase-3 proteins in group D、E、F weresignificantly less than group B、C（P＜0.01）, but more than in group A, whereas TrkAproteins is more than in group B and C（P＜0.01）.4. Retinal p75NTR mRNA in group D、E、F were significantly less than groupB、C（P＜0.01）, but more than in group A, whereas TrkA mRNA is more than in groupB and C.Part three: The effect of NGF on the human scleral fibroblast cultured in vitroMethods1. Primary culture of human scleral fibroblast was performed with sclera tissuefrom human embryonic eyes and identified with vimentin immunohistochemistystaining.2. MTT assayed the cell proliferation of scleral fibroblast at varying NGFconcentration. The cell cycle of HSF with stimulation of NGF is analyzed by the flowcytometry.3. Hydroxyproline contents （collagen synthesis） were measured with thechloramine T method.4. Statistical analysis refers to part one.Results1. The human scleral fibroblasts show good biological characteristics in primaryand secondary culture. Vimentin immunohistochemisty staining of HSF is positive.2. NGF can promote the cell proliferation of scleral fibroblast at theconcentration 25-200μg/L in dose and time dependent manner, when theconcentration of NGF is 100μg/L, the proliferative effect is the most significant（P＜0.01）. The result of flow cytometry reveals that with stimulation of NGF thepercentage of the cells in G₁ phase is decreased while the percentage of the cells in Sphase is increased and the Prl（cell proliferation index） is increased as well（P＜0.01）.3. NGF can stimulate HSF to increase the collagen protein production. Thiseffect is also in dose-depended manner. When the concentration of NGF is 50μg/L,the effect is the most powerful.Conclusions1. Form deprivation can succeed in setting up myopia model of guinea pigs,which cause the eyes axis to lengthen, induce to axis myopia;2. Investigating the dynamic expression and function of the protein and mRNAof NGF and TrkA on retina in form deprivation of guinea pigs. FDM made the expression of NGF and TrkA of protein and mRNA in retina down-regulating. NGFand TrkA may play an important role on the formation and development of myopia.3. Form deprivation in guinea pigs made apoptosis of retinal outer and innernuclear layer and the expression of p75NTR protein and Caspase-3 protein increased,whereas p75NTR mRNA increased significantly, too. The increased expression ofp75NTR and Caspase-3 in retina was consistent with retinal apoptosis.4. NGF can effectively reduce apoptosis by down-regulating of p75NTR andCaspase-3 and increased expression of TrkA. The rescue of NGF on retina was in adose dependent manner.5. NGF can stimulate the proliferation of HSF significantly and increase theproduction of collagen protein in a dose-dependent manner.更多还原