【作者】 沙倩；

【导师】 高殿文；

【作者基本信息】 中国医科大学 ， 眼科学， 2009， 博士

【摘要】 目前青光眼已被列为世界上第二位的致盲眼病,但其发病机制还不十分清楚,世界上尚无一种视神经保护药物能够通过美国的食品药品监督管理局的审核批准投入到临床使用。目前迫切的需要对青光眼视神经保护进行深入的研究,而神经免疫保护是最新提出的防止RGCs进行性丧失的策略之一。在世界范围内,青光眼视神经损害的免疫学机制及视神经保护的免疫学途径越来越受到研究者们的关注,国外的研究认为,由于自身免疫调节的紊乱,导致视网膜及视神经的某些成分改变而具有抗原性,从而引发自身免疫反应,导致视网膜及视神经损害。免疫系统在青光眼发病中所起的作用是双重的,既有神经保护作用亦有神经损害作用。更好地了解免疫系统在青光眼视神经损伤中的作用,将会帮助我们在青光眼的防治上形成一更好的防治策略。对于青光眼的病人,如何使得保护性免疫与自动免疫引起的神经退行性病变损伤相平衡将决定神经节细胞的命运。CNS通过自身的胶质细胞和浸润至CNS的效应细胞行使免疫防御功能,TLR4是一固有免疫之受体,小胶质细胞表达TLRs与其活化状态是密切相关的,能和PAMPs结合的TLRs在小胶质细胞上的表达提供了一个在病原刺激与免疫应答之间关键的联系机制,外源的病原体通过与TLRs结合启动固有免疫应答进而促进获得性免疫应答。而TLR4在青光眼视神经损伤中的具体作用机制不详。本课题拟从基因及蛋白水平上研究TLR4和其NF-kB信号转导在小胶质细胞活化中的作用及意义,利用免疫组化的方法研究TLR4和小胶质细胞在正常大鼠视网膜与慢性高眼压视网膜上的表达部位与相关性,并探讨TLR4及其NF-κB信号通路在视神经损伤中的可能的作用机制。为深入揭示TLR4与小胶质细胞的功能关系,寻找有效的TLR拮抗剂或是阻断TLR信号通路以实现最佳的小胶质细胞活化状态,为青光眼视神经损伤的免疫治疗提供理论依据。1、大鼠慢性高眼压模型的制备:烧烙3条巩膜静脉术中联合丝裂霉素的应用建立大鼠慢性高眼压模型。TONO-PENII型笔式眼压计测量术前,术后30min、2h、1d、3d、7d、14d、28d、56d眼压。2、光镜下视网膜形态学的观察:HE染色,并测量视网膜内界膜至外界膜距离即视网膜厚度。3、模型建立后2h、1d、3d、7d,、4d、28d、56d分别取5只高眼压组、假手术组及空白对照组大鼠鼻侧一半视网膜,在视网膜铺片上行免疫组织化学染色,在激光共聚焦显微镜下观察视网膜TLR4、小胶质细胞的表达情况。4、应用RT-PCR法检测各组视网膜组织中TLR4、小胶质细胞的基因mRNA表达及Western-Blot法检测各组视网膜组织中TLR4、小胶质细胞的蛋白质的表达,统计结果均以均数士标准差表示,多组均数间的比较用oneway-ANOVA分析,手术组与空白对照组均数间的比较用配对t检验分析,均用SPSS13.0软件进行统计分析。5、小胶质细胞活化模型的建立:在小胶质细胞的培养基上划痕建立的小胶质细胞活化的细胞模型,应用RT-PCR法检测各组小胶质细胞中TLR4、P50、IL-6、TNF-α的基因mRNA表达及Western-Blot法检测各组视网膜组织中TLR4、P50、IL-6、TNF-α的蛋白质的表达,并检验封闭NF-κB信号通路后各组视网膜组织中P50、IL-6、TNF-α的蛋白质及基因mRNA的表达,统计结果均以均数士标准差表示,多组均数间的比较用oneway-ANOVA分析,划痕组与对照组均数间的比较用配对t检验分析,均用SPSS13.0软件进行统计分析。结果1、大鼠慢性高眼压模型眼的术后各时间点的眼压与假手术组及空白对照组的差异具有统计学意义。2、大鼠慢性高眼压模型眼的术后各时间点的光学显微镜显示视网膜厚度经历一由厚变薄的过程。3、大鼠慢性高眼压模型眼的术后各时间点的免疫组化结果显示TLR4和小胶质细胞在视网膜组织中早期即有表达,术后7d达高峰。4、大鼠慢性高眼压模型眼的术后各时间点的Western-Blot显示TLR4和小胶质细胞在视网膜组织中早期即有表达,结果显示TLR4和小胶质细胞在时空上具有相关性。5、小胶质细胞活化的细胞模型的各实验组及各时间点的Western-Blot、RT-PCR显示TLR4、IL-6、TNF-α、P50的蛋白表达组间差异具有统计学意义。结论1、正常大鼠视网膜组织的色素上皮层可见TLR4的少量表达,并可见静态的小胶质细胞存在。2、TLR4在大鼠慢性高眼压不同时间点的视网膜上的表达,体现了其在大鼠慢性高眼压视神经退行性病变中的免疫调节作用。3、小胶质细胞的活化与TLR4在时空表达上具有相关性。4、TLR4及其信号通路在小胶质细胞的激活过程中具有重要的作用,TLR4与小胶质细胞的激活呈正反馈调节作用。更多还原

【Abstract】 Discussion of TOLL-like Receptor 4 and Its NF-κB Signal Transduction Pathway at the Inj ury and Mechanism with Chronic RatS Retinal IOP ElevationIntroductionAt present glaucoma has been listed as in the world the second blinding ophthalmopathy,but its pathogenesis also not very clear,in the world still did not have one kind of optic nerve protection medicine to be able to invest through the United States Food and Drug Surveillance Administrative bureau’s verification authorization into the clinical use.At present urgent needs to protect to glaucoma optic nerve conducts the deep research,but the nerve immunity protection is proposes most newly prevents RGCs to carry on one which of strategies the nature loses.Worldwide, glaucoma damage the optic nerve and optic nerve immunological mechanism of immunological protection by means of increasing concern to researchers,the study abroad,as a result of autoimmune disorder regulated,resulting in the retina and optic nerve changes in certain components and with antigens,thereby triggering the autoimmune response,resulting in damage to the retina and optic nerve.The immune system in the pathogenesis of glaucoma role is twofold,both have neural protective effects of nerve damage.A better understanding of the immune system in glaucoma the role of optic nerve injury,will help us in the prevention and treatment of glaucoma to create a better control strategies.For glaucoma patients,how to make a protective immune and auto-immune disease caused by neurodegenerative injury equilibrium will determine the fate of ganglion cells.CNS glial cells through its own and the effects of CNS-infiltrating cells to immune defense functions of the exercise,TLR4 is a receptor of innate immunity,TLRs expression of microglia and their activation status are closely related to the combination of TLRs and PAMPs in small Glial cells provide a stimulus in the pathogenic immune response and the links between the key mechanisms of exogenous pathogens combined with the TLRs to activate innate immune responses to promote the acquired immune response.Glaucoma and optic nerve injury in TLR4 specific mechanism is unknown.The topic to be from the level of gene and protein research TLR4 and its NF-kB signal transduction activation of microglial cells in the role and significance of the use of immunohistochemical method of TLR4 and microglial cells in the normal rat retina and chronic ocular hypertension on the expression of retinal location and relevance,and to explore the TLR4 and NF-κB signaling pathway in the optic nerve injury in the mechanisms.For an in-depth TLR4 revealed microglia cells and functional relationships,to find effective antagonists of TLR or TLR signaling pathway blocked in order to achieve the best possible state of microglial cell activation, for glaucoma optic nerve damage and provide a theoretical basis for immunotherapy.Materials and methods1.Rat chronic high intraocular pressure model preparation:In the cauterize 3 venae sclerales technique unites the silk crack mildew element the application to establish the big mouse chronic high intraocular pressure model.Before TONO a PENII pen type eye pressure meter photomicrometrology,after the technique,30min、2h、1d、3d、7d、14d、28d、56d intraocular pressure.2.Under light microscope retina morphology observation:HE dyes,and surveys in the retina the membrane to the outside membrane distance is retina thickness.3.After model building 2h、1d、3d、7d、14d、28d、56d take 5 high intraocular pressure groups,the sham-operation group and the blank control group big mouse nose side half retina separately,dyes in the retina shop piece upward immunity histochemistry,in the laser altogether focuses under the microscope to observe retina TLR4,the small spongiocyte’s expression situation.4.Law examines in each group of retina organization using RT a PCR TLR4,the small spongiocyte’s gene mRNA expression and Western a Blot law examines in each group of retina organization TLR4,the small spongiocyte’s protein expression,the statistical result indicated by the mean value gentleman standard deviation that during many group of mean value’s comparisons with oneway a ANOVA analysis,the surgery group and the blank control group mean value’s comparison with pair the T-test analysis,uses the SPSS 13.O software to carry on the statistical analysis. 5.Small spongiocyte activation model establishment:Cell model which the scratch establishment’s small spongiocyte activates on the small spongiocyte’s culture medium,law examines in each group of small spongiocyte using RT-PCR TLR4,P50, IL-6,the TNF-αgene mRNA expression and Western a Blot law examines in each group of retina organization TLR4,P50,IL-6,the TNF-αprotein expression,the statistical result indicated by the mean value gentleman standard deviation,during many group of mean value’s comparisons with oneway a AN OVA analysis,the scratch group and the control group mean value’s comparison with pair the T-test analysis,uses the SPSS 13.0 software to carry on the statistical analysis.Results1.Rat model of chronic high intraocular pressure Preparation:The burning branded three episcleral veins on intraoperative application of mitomycin United set up rat model of chronic high intraocular pressure.TONO type 1 PENll pen tonometer measuring preoperative and postoperative 30min,2h,1d,3d,7d,14d,28d,56d of intraocular pressure.2.Light microscopy observation of retinal morphology:HE staining,and measurement of retinal internal limiting membrane to the outside world that the retinal thickness from the film.3.Model set up after 2h、12h、24h、1w、4w、8wrespectively five high-tension group, sham-operated group and the normal rat retina at the nasal half of the retina chip up Shop immunohistochemical staining,laser at retina was observed under confocal microscope TLR4,a small expression of glial cell line.4.The application of RT-PCR assay in retinal tissue of each group TLR4, microglial cells express the gene mRNA and Western First Blot assay in retinal tissue of each group TLR4,microglial cells express the protein,are both results standard deviation of the number of people said that many groups were compared between the number one ANOVA using oneway analysis,drug group and the number of non-treatment group comparisons were paired t test analysis with all SPSS13.0software used for statistical analysis.5.Microglial cell activation model:RT-PCR Detection of microglial cells in each group of TLR4,P50,IL-6,TNF-a gene mRNA expression and Western First Blot assay in retinal tissue of each group NF-kB,P50,IL-6,TNF-a protein expression results are in both the standard deviation of the number of people said that many groups were compared between the number one ANOVA using oneway analysis of the treatment group and non-treatment group mean comparison between the analysis of paired t tests were used for statistical analysis software SPSS1 3.0.Conclusion1.Normal rat retinal pigment epithelium organizations shows that a small amount of TLR4 expression,and can be seen static microglia cells.2.TLR4 chronic high intraocular pressure in rats at different time points of the retina expresses its chronic high intraocular pressure in the rat optic nerve degeneration in the role of immune regulation.3.The activation of microglial cells and TLR4 expression in space and time relevant.4.TLR4 and its signaling pathway NF-κB microglial cells in the activation process plays an important role,TLR4 and the activation of microglial cells positive feedback regulation.更多还原