【作者】 赵同涛；

【导师】 阴正勤；

【作者基本信息】 第三军医大学 ， 眼科学， 2008， 博士

【摘要】 视网膜退行性变疾病是严重的致盲眼病,对于这类疾病目前尚缺乏有效阻止病变进展和恢复视网膜功能的治疗方法。其中,视网膜色素变性(Retinitis pigmentosa,RP)是一组以进行性感光细胞及色素上皮功能丧失为共同表现的遗传性疾病,是遗传性视觉损害和盲目的最常见原因之一。近年来对视网膜色素变性疾病的临床及基础研究取得一定进展,但视网膜色素变性的发病机制,及在病变过程中各种视网膜神经细胞的病理改变及功能状态尚不清楚。Müller细胞是脊椎动物视网膜中一种主要的胶质细胞,在功能上起到联系各种视网膜神经细胞的作用,是视网膜各种细胞之间物质交换的结构基础。在正常视网膜中具有参与视网膜糖代谢、调节视网膜血流、参与神经递质循环、调节视网膜水电解质平衡、参与感光色素循环过程及控制神经元兴奋性等功能。在视网膜的各种病理条件下,Müller细胞会发生活化,在形态、数量及蛋白表达上均发生一系列变化。研究表明,在视网膜脱离、增殖性玻璃体视网膜病变(PVR)和增殖性糖尿病视网膜病变(PDR)中Müller细胞本身变得肥大同时也发生了增殖及纤维化。在一定条件下,成熟Müller细胞还具有分化的潜能。研究表明在急性视网膜损伤模型中,成熟Müller细胞在增殖的同时还能表达PAX6、Nestin等视网膜干细胞的标志物。体外研究也表明,在一定条件下,Müller细胞可以分化为视网膜干细胞。此外,Müller细胞发生活化后还有一个显著特点就是细胞电生理特性发生了变化。Müller细胞不同于其他视网膜神经细胞的特点是其细胞膜上具有大量的K+通道,这与其缓冲视网膜组织内K+及参与神经递质循环的功能有关。研究表明,在视网膜脱离及急性视网膜损伤等病理模型中,Müller细胞在活化的同时均表现出以K+电流为主的电生理特性的改变,这种离子电流的改变可以反应出Müller细胞的功能及分化状态。对于Müller细胞活化的机制,目前尚不清楚。细胞外信号调节激酶(extracellular signal - regulated kinase , ERK)介导的信号传递途径是调节细胞生长、发育及分裂的信号网络的核心,众多研究表明嘌呤类受体P2Y-ERK信号通路与星形胶质细胞的增殖分化密切相关,因此也可能参与调节了Müller细胞的活化过程。RP作为一种慢性病变刺激是否也会导致Müller细胞活化,目前尚未见报道。本实验室前期采用皇家外科学院(Royal college of surgeons,RCS)大鼠对这一问题进行了初步研究。发现,RCS鼠视网膜色素变性过程中,Müller细胞能形成所谓“胶质封闭”(glial seal),阻断了视网膜神经上皮与色素上皮之间的联系,这说明在RP中,Müller细胞有可能发生了增殖及迁移。本实验室对RCS鼠进行蛋白印迹研究还发现,RCS鼠在视网膜变性过程中,其视网膜组织中视网膜干细胞标志物CHX10的表达呈上升趋势。此外,在我们实验室前期对RCS鼠视网膜神经细胞电生理的研究中发现神经节细胞、双极细胞等都发生了离子电流的改变,那么Müller细胞作为视网膜神经细胞之间联系的桥梁,其离子电流特性也可能发生了改变。因此本研究提出假设,在视网膜色素变性病变发生发展过程中,视网膜色素变性作为一种慢性刺激导致了Müller细胞的活化,表现为增殖、分化及离子电流改变等一系列与活化相关的病理生理改变,而P2Y1受体-ERK信号通路可能参与了Müller细胞活化的调节过程。针对上述假说,本文的主要研究内容及结果如下:1、不同发育阶段RCS鼠视网膜Müller细胞活化状态的研究。采用冰冻切片、免疫组织化学方法特异性标记RCS鼠视网膜Müller细胞,研究在视网膜变性发展过程中,Müller细胞活化的病理改变。结果发现:1)以上结果提示,RCS鼠视网膜Müller细胞相对于对照组发育较快,而且随着病程的进展,Müller细胞逐渐变得肥大、排列开始紊乱,并在视网膜下腔局部形成纤维层。提示RCS鼠视网膜Müller细胞发生了明显的病理改变。2)对不同阶段RCS鼠视网膜Müller细胞进行计数后发现,在P30d,两组视网膜Müller细胞数量无显著性差异(P>0.05);而在P60d、90d、120d,RCS鼠视网膜Müller细胞数量均显著高于对照组(P<0.05或P<0.01),说明在RCS鼠视网膜变性的中晚期,Müller细胞数量明显高于对照组,说明Müller细胞在视网膜变性的中晚期发生了明显的增殖;3)在P30d、60d、120d时RCS鼠Müller细胞GAFP表达明显高于对照组,提示在视网膜变性开始后Müller细胞发生了活化。RCS鼠视网膜变性开始后,Müller细胞ERK蛋白表达量明显高于对照组,进一步证实Müller细胞活化过程中伴有ERK的激活。4)由此可见,RCS鼠视网膜变性过程中Müller细胞可以表达视网膜干细胞标志物CHX10,说明视网膜变性过程中成熟Müller细胞在活化的同时可以向视网膜干细胞等其他神经细胞方向分化。2、RCS鼠视网膜变性过程中Müller细胞活化的离子通道特性研究。采用急性分离的视网膜Müller细胞膜片钳记录的方法,对不同发育阶段的RCS鼠Müller细胞进行全细胞记录,研究Müller细胞在视网膜变性发展过程中基本膜学特性、内外向K+电流的性质及大小的变化。结果发现:1)RCS鼠视网膜变性过程中Müller细胞的静息膜电位逐渐降低,输入阻逐渐升高,并且都与对照组有显著性差异,说明RCS鼠视网膜Müller细胞膜逐渐去极化。RCS鼠视网膜变性过程中Müller细胞的膜电容逐渐增加,并且显著高于对照组,说明RCS鼠视网膜Müller细胞膜逐渐增大,2)在给予超极化和去极化脉冲时,Müller细胞主要表现出内向及外向的K+电流,能够被TEA及Ba2+阻断。说明正常Müller细胞膜上离子通道以K+通道为主,并且具有内向整流特性,这符合Müller细胞缓冲视网膜K+的功能特点。3)在视网膜变性早期,Müller细胞内向及外向K+电流均增大,加速清除视网膜组织内由于兴奋性毒性产生的K+,从而对视网膜神经细胞起到一定保护作用;而晚期,Müller细胞清除K+能力则大大降低,加重视网膜各种神经细胞的损害。4)RCS鼠P30d时K+电流密度显著高于对照组(P<0.05),P90d时K+电流密度显著低对照组(P<0.05)。提示随视网膜色素变性进展,Müller细胞膜上单位面积K+电流减少,表现出类似原始Müller细胞(或干细胞)K+电流的特征,说明其K+通道类型可能发生改变。5)RCS鼠Müller细胞K+通道I-V曲线的变化同样符合其离子电流特性,即在视网膜变性早期Müller细胞缓冲K+能力增强,因而相对于对照组,随着膜电位的去极化,细胞膜K+电流升高更加明显;而在视网膜变性的晚期,相对于对照组,RCS鼠Müller细胞随着膜电位的去极化,K+电流却发生了更显著的降低。3、RCS鼠变性视网膜对正常视网膜Müller细胞活化的机制研究。采用正常Müller细胞与RCS鼠视网膜混合细胞共培养的方法,检测Müller细胞的增殖分化改变。同时采用western-blot、RT-PCR等方法检测在共培养系统中, P2Y1受体-ERK信号通路的表达情况。共培养分组:1)正常Müller细胞与P30d之RCS鼠视网膜混合细胞共培养,为实验组;2)正常Müller细胞与P30d之RCS鼠视网膜混合细胞共培养,同时在培养系统中加入P2Y1-ERK通路阻断剂PD98059(20μM),为阻断组;3)正常Müller细胞与P30d之对照组大鼠视网膜混合细胞共培养,为对照组。结果发现:1)提示共培养3d、7d时Müller细胞在变性视网膜混合细胞的作用下发生增殖。在变性视网膜混合细胞作用下,正常Müller细胞的GFAP和ERK蛋白表达均高于对照组,说明变性视网膜可以使正常Müller细胞发生活化。在变性视网膜组织与正常Müller细胞共培养一定时间后,正常Müller细胞可能发生了一定程度的分化,表达视网膜干细胞标志物。2)共培养1d时,实验组与加入阻断组的Müller细胞增殖率无显著性差异(P>0.05);共培养3d、7d时实验组Müller细胞增殖率明显高于阻断剂PD98059组(P<0.05);提示变性视网膜混合细胞促进正常Müller细胞增殖的作用能够被P2Y1-ERK信号通路阻断剂PD98059阻断。3)PD98059能够阻断Müller细胞的活化并使Müller细胞表达GFAP及ERK蛋白减少。RT-PCR结果共培养3d及7d时实验组P2Y1受体mRNA表达增加,并高于阻断组。因此,我们得出以下结论:1、RCS鼠视网膜色素变性过程中Müller细胞发生了活化,表现为细胞本身的肥大、局部胶质封闭的形成及细胞发生的明显增殖,随着视网膜变性的进展,Müller细胞活化更加明显。而正常Müller细胞在RCS鼠视网膜混合细胞作用下也可以发生增殖反应。2、在体及离体实验都说明,RCS鼠视网膜素色变性可以使Müller细胞GFAP和ERK蛋白表达的增加,这从蛋白水平进一步说明Müller细胞在视网膜色素变性过程中发生了活化反应;同时,Müller细胞可以表达视网膜干细胞标志物CHX10,说明Müller细胞活化以后可能发生了向视网膜干细胞方向的分化。3、RCS鼠视网膜色素变性的中晚期,Müller细胞静息膜电位降低,膜电容、输入阻抗增大,这与Müller细胞在视网膜色素变性过程中增生、肥大的病理改变改变相一致。4、RCS鼠视网膜Müller细胞的离子通道特性主要表现为去极化时的外向K+电流和超极化时的内向整流K+电流。在视网膜变性早期,内外向电流幅值均高于对照组,而在变性晚期则显著低于对照组,这揭示了Müller细胞在视网膜变性过程中功能状态的变化,即早期缓冲K+能力增强对变性视网膜中K+造成的神经毒性起到一定修复作用,而晚期缓冲K+能力降低,造成视网膜中K+堆积,加重视网膜神经细胞损害。5、发现随视网膜色素变性进展,RCS鼠Müller细胞膜上单位面积K+电流减少,说明Müller细胞K+通道类型可能发生改变、数量可能减少,这中表现类似于原始Müller细胞(或干细胞)K+电流的特征,因此从电生理方面进一步证实Müller细胞可能发生了分化。6、Müller细胞发生活化的同时伴有ERK蛋白和P2Y1受体mRNA表达的增加,而当加入ERK信号通路阻断剂后,Müller细胞的活化反应被抑制,同时ERK蛋白表达及P2Y1受体mRNA含量明显降低。这说明,RCS鼠视网膜色素变性过程中Müller细胞的活化反应可能由P2Y1受体-ERK信号通路所介导。更多还原

【Abstract】 Retinal degeneration (RD) is a group of severe diseases leading to blindness, which lack effective therapeutic measures. Among the RD, Retinitis pigmentosa (RP) is the most common retinal inherited disease, which featured in a progressing apoptosis of photoreceptors and disfunction of retinal pigmented epitheliums (RPE). The pathological mechanism and functional changes of neurons in the progress of RP is still unknown.Müller cells are main glial cells in the mammalian retina. They connect almost all the retinal neurons by their specialized morphology, which is the foundation of normal retinal function. Müller cells are involved in many retinal physiological activities including glycometabolism, blood regulation, neurotransmitter cycling, homeostasis and control of neuronal excitability.Under pathological conditions, Müller cells can be activated to perform a series of changes in morphology, amount and protein expression. It was reported that Müller cells could proliferate dramatically, becoming hypertrophy and fibrosis in retina detachment, PVR and PDR. Besides, the mature Müller cells may re-enter the cell cycling, and differentiate into retinal stem cells expressing PAX6, Nestin in some acute retinal injuries.Meanwhile, Müller cells showed obvious changes of the electrophysiological properties when being activated. The characteristic electrophysiological properties of Müller cells distinguished to other retinal cells are large amounts of potassium channels in the membrane, which is related to Müller cells’function of buffering K+. In retinal detachment and acute retinal injuries, K+ channels currents of Müller cells decreased significantly.The mechanism of Müller cells activation is still not clear. Extracellular signal - regulated kinase (ERK) signal pathway is the core in regulation of cells’growth, development and mitosis. Many articles reported that purinergic receptor P2Y1-ERK signal pathway was closely related to the proliferation and differentiation of astrocytes. So the P2Y1-ERK pathway is probably involved in the activation of Müller cells . As a chronic inflammatory disease, if RP can also activate Müller cells , and how Müller cells change in RP is still unknown.Our previous studies showed that Müller cells glial seal formed in the subretinal space in the progress of RCS rats’retinal degenetation, which is considered to block the connection between neuroretinal epitheliums and RPE.。Quantitive test of RCS retinal proteins showed, CHX10, a marker of retinal stem cells , increased significantly during the progress of retinal degeneration. Our patch clamp recording on RCS rats retinal neurons showed that a dramatically changes of ionic currents occurred in the ganglion cells and the bipolar cells. So as a bridge among retinal neurons, Müller cells’ionic currents may also changed in progress of RCS rats retinal degeneration.Based on above, we hypothesis that Müller cells also can be activated in the progress of RP to proliferate and re-differentiate, and display changes of ionic currents cross the membrane. P2Y1-ERK signal pathway may mediate the activation of Müller cells .Our main research contents and results are showed as following:1、Activated state of Müller cells in RCS rats at different development stage.Methods:frozen section, immunohistochemistry . Results:1)Müller cells of RCS rats developed more rapidly than control, and became hypertrophy , disorder. And a local fibril layer former in the subretinal space. 2)At P60d、90d、120d,amount of Müller cells in RCS rats retina was significantly higher than control(P<0.05 or P<0.01) which implied that Müller cells of RCS rats proliferated dramatically. 3)At P30d、60d、120d, the expression of GFAP and ERK in RCS rats retina was significantly higher than control ,which implied Müller cells were activated , and ERK might be involved in it. 4)Müller cells of RCS retina could express CHX10, a maker of retinal stem cells, which proved that differentiation of Müller cells happened in the progress of retinal degeneration.2、Properties of K+channels currents in Müller cells from RCS retina.Methods: patch clamp recording of the acute isolated Müller cells. Results:1)With theprogress of RD, RMP of Müller cells from RCS rats retina depolarized dramatically, and IR, Cm increased significantly higher than control (P<0.05), implying that Müller cells became hypertrophy in responds to RD.2)inward and outward currents were recorded when giving hyper- and depolarized voltage steps, which could be blocked by Ba2+ and TEA, implying a predominant distribution of K+ channels on Müller cells . 3)at the early stage of RCS rats retinal degeneration, the inward and outward currents increased, and were significantly higher than control, which is considered to eliminate the extracelluar K+ produced in the RD, to be helpful to neuron survival. At late stage, the inward and outward currents decreased, and were significantly lower than control, which worsened the retinal impairments. 4)At P30d the K+ currents density of Müller cells in RCS rats was significantly higher (P<0.05) than control; At P90d, the K+ currents density of Müller cells in RCS rats was significantly lower (P<0.05). The decease of K+ current density implied that the number of K+ channels might deceased during retinal degeneration. 5)The I-V curves of RCS rats’Müller cells showed, at P30d, with the increase of voltage impulse, the current amplitude of RCS rats Müller cells increased more rapidly than control, and both inward and outward current amplitudes were higher. At P90d, it changed adversely.3、Effect of RCS rats retinal mixed cells on the normal cultured Müller cells, and P2Y1-ERK signal pathway’s involvement in it.Methods: cocluture of normal cultured Müller cells and RCS rats retinal mixed cells, immunohistochemistry, western-blot、RT-PCR. Results: 1)At 3d、7d after coculture, the normal Müller cells proliferated dramatically, and the expression of GFAP and ERK was significantly higher than control. Part of normal Müller cells expressed CHX10, which implying that normal Müller cells were activated by the RCS rats retinal mixed cells. 2)At 3d、7d after coculture, the proliferation rate of normal Müller cells increased dramatically(P<0.05), which could be completely blocked by PD98059, the blocker of ERK pathway. It is further proved that ERK pathway might mediate the activation o Müller cells . 3)The expression of GFAP and ERK was significantly decreased after blocking the ERK pathway by PD98059. RT-PCR results showed , at 3d、7d after coculture, the quantity of normal Müller cells’P2Y1 mRNA increased , and significantly higher than control and PD98059 group.Based on the results, we concluded:1、Müller cells could be activated during the progress of RCS rats’retinal degeneration. After being activated, Müller cells begin to proliferate, becoming hypertropy, and formed local glial seal in the subretinal space. The expression of GFAP increased with progress of retinal degeneration. 2、After being activated, Müller cells might differentiate into retinal stem cells in responds to activation by expressing CHX10. 3、At middle and late stage of retinal degeneration, RMP of RCS rats’Müller cells became more depolarized, the IR and Cm of RCS rats’Müller cells increased dramatically, which provided electrophysiological evidence to Müller cells activation. 4、At the early stage of RCS rats retinal degeneration, Müller cells enhanced their ability of buffering K+ by increasing the trans-membrane K+ currents. At late stage, trans-membrane K+ currents of RCS rats’Müller cells decreased significantly, implying a disorder of Müller cells function, which might worsen the retinal impairments. 5、Coincident with the change of K+ currents, the decease of K+ current density implied that the number of K+ channels might deceased during retinal degeneration. 6、In RCS rats, the expression of ERK in Müller cells increased with the progress of retinal degeneration, and quantity of P2Y1 mRNA increased under the stimulation of RCS rats’retinal mixed cells. So the P2Y1-ERK signal pathway might mediate the activation of Müller cells.更多还原