青光眼神经节细胞退行性病变相关研究

发布时间：2018-05-17 04:33

本文选题：青光眼 + 神经退行性病变　；参考：《华中科技大学》2013年博士论文

【摘要】：青光眼是一种复杂的致盲性神经退行性疾病。青光眼的神经退行性病变发生在视网膜神经节细胞及视神经轴突,涉及先天遗传倾向,后天危险因素以及与年龄增长相关的环境刺激。眼内压升高被认为是青光眼疾病发生发展的重要危险因素之一,也是目前可以干预的因素。至今青光眼的治疗还是基于降低眼压。近年来研究发现,除了眼压升高,在青光眼中多种刺激因素影响了细胞内环境稳定,而且神经元对刺激因素的抵抗力也决定了其内在易感性。有证据显示当视网膜神经节细胞暴露在青光眼性压力刺激下,导致其出现一系列相关联的细胞下游通路活动,包括线粒体功能异常,蛋白水解级联反应,内质网压力和氧化应激等。引起神经起始损伤和青光眼性神经退行性病变的具体分子机制一直在被广泛研究并存在很大争议。青光眼神经节细胞损伤的因素有很多,目前有很多学说试图解释青光眼的发病机制,如机械压力学说、氧化应激学说等,而最近研究的热点是青光眼的自身免疫和异常蛋白沉淀这两大方面：研究发现青光眼神经退行性病变涉及免疫系统,包括天然免疫和获得免疫在内的不同成分,还有越来越多的证据将青光眼的发病机制指向补体系统的激活,补体激活的产物,如Clq, C3和膜攻击复合物(MAC)在青光眼模型动物和青光眼病人的视网膜中被发现。本研究的前两个部分试图探讨补体系统的激活在青光眼发病机制中所扮演的角色。第一部分为体内研究,连续观察补体系统相关蛋白在大鼠青光眼模型中的变化。第二部分为体外研究,探讨补体系统激活终产物膜攻击复合物C5b-9对视网膜神经节细胞凋亡和程序化坏死的影响。另一方面,神经退行性病变是指神经元细胞在结构及功能上的逐渐损失,最终死亡的病变,其发病机理涉及蛋白的异常加工,错误折叠以及在蛋白在细胞内外的异常聚集。视网膜也是中枢神经系统的一部分,因此在神经退行性病变的进程中,视网膜和视神经会受到相似的影响。异常蛋白在神经元的沉积,包括Ap和异常Tau蛋白的沉积,是神经节细胞另一损伤因素。本研究第三部分是观察Tau蛋白在青光眼视网膜中的变化并探讨氯化锂在慢性青光眼神经退行性病变的作用及治疗机制。第一部分：大鼠青光眼模型中补体系统相关蛋白的变化目的：研究补体系统的激活对慢性青光眼高眼压模型中视网膜神经节细胞的影响。方法：用DA大鼠构建慢性高眼压模型,将动物分成4组,每组5只,左眼为手术眼,通过往巩膜静脉注射高渗盐水的方法提高眼内压,右眼不做处理作为正常对照眼。动物自手术后1周,3周,8周和16周分别处死,免疫荧光法测试高眼压眼和对照眼视网膜内的膜攻击复合物(MAC, C5b-9), C3和衰变加速因子(DAF)的表达水平。结果：C5b-9自青光眼大鼠眼压升高8周起表达明显上调,C3的表达在高眼压中也有所提高。而衰变加速因子DAF的表达发现在8周和16周高眼压组明显降低。结论：在慢性青光眼动物模型视网膜节细胞层补体活动产物上调和补体调节蛋白衰变加速因子DAF的下降提示补体激活和补体调节的失衡,此现象是否在青光眼的发病机制中扮演潜在作用还需进一步研究。第二部分：膜攻击复合物C5b-9对视网膜神经节细胞的凋亡和程序化坏死的影响目的：视网膜神经节细胞的损失是青光眼的特征,但是神经节细胞死亡的调节机制仍未研究清楚。本部分的研究目的是确定膜攻击复合物C5b-9是否诱导神经节细胞的死亡和／或者调节了视网膜神经节细胞对其他细胞压力作用的敏感性。方法：本研究使用RGC-5细胞系为研究对象,用抗CD59抗体阻断CD59后, RGC-5细胞作用于正常人血清,用免疫荧光染色法观察细胞形态学,流式细胞技术以及用PARP剪切和活化的caspase-3作为探针的western blot技术检测细胞凋亡的程度。同时用程序化坏死抑制剂AGK2作用于细胞,用流式细胞技术检测细胞程序性坏死的程度。在有或没C5b-9生成的条件下,ionomycin, staurosporine,双氧化氢和chelerythrine对RGC-5细胞的敏感性的变化也被观察。结果：在暴露于C5b-9的条件下,RGC-5细胞出现凋亡。RGC-5细胞还在staurosporine作用后出现凋亡,但对ionomycin和过氧化氢存在抵抗作用。C5b-9能够明显增加staurosporine引发RGC-5细胞凋亡和程序化坏死的敏感度。结论：低水平的C5b-9可以引起RGC-5细胞的凋亡,应用不同的激动剂证实C5b-9能特异地敏感化一些凋亡和程序化坏死的通路。研究结果揭示了补体系统在视网膜神经节细胞损失中的潜在作用。第三部分：Tau蛋白在鼠慢性青光眼模型中的变化和机制研究目的：研究在慢性青光眼性损害时视网膜神经节细胞层中Tau蛋白磷酸化的变化,并通过利用氯化锂抑制Tau上游激酶GSK-3β活性的实验,为在临床上用氯化锂治疗慢性青光眼神经退行性病变提供实验依据。方法：建立慢性高眼压SD大鼠模型,分为两组各10只。一组为高眼压未治疗组,右眼为高眼压模型眼,左眼为正常对照眼；另一组为高眼压氯化锂治疗组,治疗组于建模当天起每日腹腔注射0.6M氯化锂(GSK-3β抑制剂)。在第2周和第4周从两组中各取5只大鼠处死并摘取眼球,用免疫荧光和Western blot观察视网膜总Tau和磷酸化Tau蛋白的变化。结果：1)高眼压模型视网膜Tau蛋白及其磷酸化水平发生了改变：总Tau含量在第2周时下降为对照眼的77.3%,在第4周时下降为对照组的60.4%; p-Tau与总Tau的比值,在第2周时几乎没有变化,在第4周时增加到对照眼的135.4%,磷酸化明显增加。2)氯化锂对高眼压模型鼠的影响：在第4周时,氯化锂治疗组视网膜总Tau含量较未治疗组明显升高,为对照眼的99%;p-Tau与总Tau的比值较未治疗组明显下降,几乎降到对照眼水平。结论：持续高眼压刺激导致大鼠视网膜总Tau蛋白表达量降低,Tau蛋白磷酸化水平增加；氯化锂能减轻由持续高眼压引起的Tau蛋白的过度磷酸化,其机制还需进一步研究。研究总结一、主要研究结果 1.在慢性高眼压模型鼠中,膜攻击复合物C5b-9在视网膜神经节细胞层的表达在眼压升高第8周明显上升,补体激活途径枢纽蛋白C3在慢性高眼压模型中的表达也上调。补体调节蛋白衰变加速因子(DAF)的表达在高眼压第8周明显下降,与C5b-9的表达变化相对应。 2.体外研究发现膜攻击复合物C5b-9可在RGC-5细胞上被诱导生成。C5b-9的生成可诱导RGC-5细胞的发生凋亡。C5b-9能够明显增加staurosporine引发细胞凋亡和程序化坏死的敏感度。 3.在慢性高眼压模型鼠中,总Tau含量在高眼压第2周时已下降,在第4周进一步降低,在第4周Tau蛋白的磷酸化明显增加；氯化锂治疗组在第4周时视网膜总Tau含量较未治疗组明显升高,磷酸化程度较未治疗组明显下降。二、研究结论 1.在慢性青光眼动物模型中,视网膜节细胞层各补体活动产物上调和补体条件蛋白DAF的下降提示补体激活和补体调节的失衡可能在青光眼的发病机制中扮演潜在作用。 2.低水平的C5b-9可以引起RGC-5细胞的凋亡,并通过多种不同的激动剂的运用,进一步证实了C5b-9能特异地使细胞的一些凋亡和程序化坏死的通路敏感化。 3.在慢性青光眼动物模型中,持续高眼压刺激导致大鼠视网膜总Tau蛋白表达量降低,Tau蛋白磷酸化水平增加；氯化锂能减轻由持续高眼压引起的Tau蛋白的过度磷酸化。
[Abstract]:Glaucoma is a complex, blinding neurodegenerative disease. Neurodegenerative diseases of glaucoma occur in retinal ganglion cells and optic axons, involving congenital genetic tendencies, acquired risk factors and environmental stimuli associated with age growth. Elevated intraocular pressure is recognized as an important risk for the development of glaucoma. One factor, it is also a factor that can be used to intervene now. The treatment of glaucoma is still based on the reduction of intraocular pressure. In recent years, it has been found that in addition to increased intraocular pressure, a variety of stimuli in glaucoma affect the stability of the intracellular environment, and the resistance of neurons to stimulating factors also determines its intrinsic susceptibility. Membrane ganglion cells are exposed to glaucoma pressure stimulation, resulting in a series of associated downstream pathway activities, including mitochondrial dysfunction, proteolysis cascade, endoplasmic reticulum pressure and oxidative stress. The specific molecular mechanisms that cause nerve initial damage and glaucomatous degenerative diseases have been widely used. There is a lot of controversy in the pan study.
There are many factors for the injury of ganglion cells in glaucoma. There are many theories to explain the pathogenesis of glaucoma, such as the theory of mechanical stress, the theory of oxidative stress and so on. The recent research focuses on the two major aspects of the autoimmune and abnormal protein precipitation of glaucoma. The study found that the neurodegenerative diseases of glaucoma involve the immune system. There is a growing number of evidence to point the pathogenesis of glaucoma to the activation of the complement system. The products activated by complement, such as Clq, C3 and membrane attack complex (MAC), are found in the retina of glaucoma model animals and glaucoma patients. The first two parts of this study attempt To explore the role of complement system activation in the pathogenesis of glaucoma. The first part is in vivo study to observe the changes of complement system related proteins in the rat model of glaucoma. The second part is in vitro study to explore the apoptosis and process of retinal ganglion cells by the complement system activation terminal product membrane attack complex C5b-9. On the other hand, neurodegenerative disease refers to the gradual loss of neuronal cells in structure and function, the eventual death of the lesion, and its pathogenesis involves abnormal processing of proteins, misfolding and abnormal aggregation of proteins in and out of cells. The retina is part of the central nervous system and therefore is in the nerve. In the process of degenerative disease, the retina and optic nerve are affected by similar effects. Abnormal protein deposition in neurons, including the deposition of Ap and abnormal Tau protein, is another damage factor of the ganglion cells. The third part of this study was to observe the changes in the Tau protein in the glaucoma retina and to explore the retrogression of lithium chloride in the chronic glaucoma. The role and therapeutic mechanism of progressive lesions.
Part one: changes of complement system related proteins in rat glaucoma models
Objective: To study the effect of complement system activation on retinal ganglion cells in chronic glaucoma with high intraocular pressure.
Methods: the model of chronic ocular hypertension was constructed in DA rats. The animals were divided into 4 groups, 5 in each group, and the left eye was the operation eye. The intraocular pressure was improved by injection of hypertonic saline into the scleral vein. The right eye was not treated as the normal eye. The animals were killed at 1, 3, 8 and 16 weeks after the operation. High intraocular pressure and control were tested by immunofluorescence. The retinal inner membrane attacks the expression levels of MAC (C5b-9), C3 and decay accelerating factor (DAF).
Results: the expression of C5b-9 was up obviously up to 8 weeks from the increase of intraocular pressure in glaucoma rats, and the expression of C3 increased in high intraocular pressure. The expression of the decay accelerating factor DAF was obviously decreased in the high intraocular pressure group at 8 and 16 weeks.
Conclusion: the up-regulation of complement activity products and the decline of complement regulatory protein decay acceleration factor DAF in the retinal ganglion cell layer of chronic glaucoma animal models suggest the imbalance of complement activation and complement regulation. It is still necessary to study the potential role of this phenomenon in the pathogenesis of glaucoma.
The second part: effect of membrane attack complex C5b-9 on apoptosis and programmed necrosis of retinal ganglion cells.
Objective: the loss of retinal ganglion cells is a characteristic of glaucoma, but the mechanism of the regulation of ganglion cell death is still not clear. The purpose of this part is to determine whether the membrane attack complex C5b-9 induces the death of ganglion cells and / or regulates the sensitivity of the retinal ganglion cells to the pressure of other cells. Sex.
Methods: in this study, the RGC-5 cell line was used as the research object. After blocking the CD59 with anti CD59 antibody, the RGC-5 cells acted on the normal human serum. The cell morphology was observed by immunofluorescence staining, the flow cytometry and the Western blot technology with PARP and activated caspase-3 as the probe were used to detect the degree of apoptosis. Sequence necrosis inhibitor AGK2 acts on cells, and the degree of programmed cell necrosis is detected by flow cytometry. The changes in sensitivity of ionomycin, staurosporine, hydrogen peroxide and chelerythrine to RGC-5 cells are also observed under or without C5b-9 formation.
Results: under the condition of exposure to C5b-9, apoptotic.RGC-5 cells in RGC-5 cells were also apoptotic after the action of staurosporine, but the resistance to ionomycin and hydrogen peroxide could significantly increase the sensitivity of staurosporine to apoptosis and programmed necrosis of RGC-5 cells.
Conclusion: low level of C5b-9 can induce apoptosis of RGC-5 cells. Different agonists have been used to confirm that C5b-9 can sensitize some apoptotic and programmed necrosis pathways specifically. The results reveal the potential role of complement system in retinal ganglion cell loss.
The third part: the change and mechanism of Tau protein in rat chronic glaucoma model.
Objective: To study the changes in the phosphorylation of Tau protein in the retinal ganglion cell layer of chronic glaucomatous damage, and to provide evidence for the clinical use of lithium chloride in the treatment of neurodegenerative diseases of chronic glaucoma by using lithium chloride to inhibit the activity of Tau upstream kinase GSK-3 beta.
Methods: the chronic high intraocular pressure SD rat model was established and divided into two groups of 10 rats. One group was the untreated group with high intraocular pressure, the right eye was the high intraocular pressure model eye, the left eye was normal to the eye, the other group was the high intraocular pressure lithium chloride treatment group. The treatment group Yu Jianmo was intraperitoneally injected with 0.6M lithium chloride (GSK-3 beta inhibitor) daily from the day of second and fourth weeks from two groups. 5 rats in each group were sacrificed and their eyeballs were removed. The changes of total Tau and phosphorylation of Tau protein were observed by immunofluorescence and Western blot.
Results: 1) the retinal Tau protein and its phosphorylation level in the high intraocular pressure model were changed: the total Tau content decreased to 77.3% in the control eye at second weeks and 60.4% in the control group at fourth weeks; the ratio of p-Tau to the total Tau was almost unchanged at second weeks, and increased to 135.4% of the control eye at fourth weeks, and phosphorylation significantly increased.2. The effect of lithium chloride on the high intraocular pressure model rats: at fourth weeks, the total retinal Tau content in the lithium chloride treatment group was significantly higher than that in the untreated group, which was 99% of the control eye. The ratio of p-Tau to the total Tau decreased significantly than that in the untreated group, almost to the control eye level. Conclusion: the sustained high intraocular pressure induced the decrease of the total Tau protein expression in the retina of rats. The level of Tau protein phosphorylation is increased. Lithium chloride can alleviate the excessive phosphorylation of Tau protein induced by sustained high intraocular pressure, and its mechanism needs further study.
Research Summary
First, the main research results
1. in the chronic high intraocular pressure model rats, the expression of membrane attack complex C5b-9 in the retinal ganglion cell layer increased obviously for eighth weeks, and the expression of the complement activation pathway hub protein C3 in the chronic high intraocular pressure model was also up-regulated. The expression of complement regulatory protein decay acceleration factor (DAF) decreased significantly at the high intraocular pressure for eighth weeks, and C5b-9 The change of expression is corresponding.
2. in vitro study found that membrane attack complex C5b-9 can be induced to produce.C5b-9 on RGC-5 cells and induce apoptosis of RGC-5 cells to induce apoptosis.C5b-9, which can significantly increase the sensitivity of staurosporine to cell apoptosis and programmed necrosis.
3. in the chronic high intraocular pressure model rats, the total Tau content decreased at the second week of high intraocular pressure, decreased further in the fourth week, and the phosphorylation of Tau protein increased significantly at fourth weeks, and the total Tau content in the retina group was significantly higher than that in the untreated group at fourth weeks, and the degree of phosphorylation was significantly lower than that in the untreated group.
Two, the conclusion of the study
1. in the animal model of chronic glaucoma, the up regulation of complement activity products and the decrease of complement conditional protein DAF in the retinal ganglion cell layer suggest that the imbalance of complement activation and complement regulation may play a potential role in the pathogenesis of glaucoma.
2. the low level of C5b-9 can induce apoptosis of RGC-5 cells and, through the use of a variety of different agonists, further confirmed that C5b-9 can sensitize some of the cell apoptosis and programmed necrosis in a special way.
3. in the animal model of chronic glaucoma, persistent high intraocular pressure (IOP) stimulates the decrease of the total Tau protein expression in the retina and the increased phosphorylation of Tau protein, and lithium chloride can reduce the excessive phosphorylation of Tau protein caused by persistent high intraocular pressure.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R775

【相似文献】