视网膜神经节细胞与Müller细胞共培养的方法研究

目的　建立一种简单的体外培养视网膜神经节细胞的方法。方法　采用Thy1 1单克隆抗体免疫吸附得到纯化的视网膜神经节细胞 ,将其接种于纯化培养的视网膜M櫣ｌｌｅｒ细胞上 ,在无血清培养液中共同培养。结果　视网膜神经节细胞在接种后 1d即可生长出短小的突起 ,至第 5d突起不断增长并可长出二级分支。结论　M櫣ｌｌｅｒ细胞在无血清培养液中 ,对体外培养的神经节细胞具有很好的支持营养作用。     

兔眼视网膜铁锈症中Müller氏细胞的变化

在电镜下观察了兔眼视网膜铁锈症中Müller氏细胞的变化。其变化有:(1)细胞变形,极性特征消失;(2)胞突扩张,填补细胞变性的空隙;(3)胞浆内出现大量线粒体;(4)并见色素颗粒及感光细胞的残片;(5)在视网膜不同层次,Müller氏细胞之间形成排列紊乱的粘着小带。证实Müller氏细胞具有相当活跃的吞噬及修补功能。推测含铁物质的毒性可能通过Müller氏细胞传递,影响视网膜色素上皮细胞及感光细胞。     

兔眼钝挫伤后视网膜 Müller细胞GFAP表达的变化

目的:探讨眼球钝挫伤后视网膜Müller细胞表达GFAP的变化规律。方法:20只兔子40眼随机分为正常对照组、挫伤组,以 3J 能量自由落体的方式制作兔眼挫伤性视网膜病变模型,于不同时段将动物麻醉致死,摘除眼球,制成病理切片用于GFAP免疫组织化学染色。图像分析系统测量视网膜GFAP表达阳性率与灰度值,并应用SPSS 12.0软件包进行统计学分析。结果:挫伤组和正常对照组视网膜内界膜下可见少许棕色阳性GFAP着色。挫伤组伤后1d,GFAP的阳性表达开始加强,随伤后时间的推移,GFAP的免疫染色已从内界膜向神经纤维层、节细胞层、内丛状层、外核层,直至神经上皮层下发展,并呈现着色加深的强阳性表达。GFAP表达的平均灰度值的统计学分析结果也显示:挫伤组和正常对照组存在统计学差异（P＜0.05）。结论:眼球钝挫伤后视网膜Müller细胞GFAP的表达持续增强,早期Müller细胞反应性胶质化对视网膜损伤有修复作用。     

黄连素对高糖培养下大鼠视网膜Müller细胞增殖的影响

目的：研究黄连素对体外高糖环境下的SD大鼠视网膜Müller细胞增殖的影响。

方法：采用酶消化法原代培养SD大鼠视网膜Müller细胞,将第2代Müller细胞随机分为正常糖浓度(5mmol/L)组、高糖浓度(25mmol/L)组、高糖+不同浓度(5、10、25、50、100μmol/L)黄连素组,分别培养24、48、72h后,采用CCK-8法检测细胞增殖活性。

结果：培养24、48、72h时,高糖浓度组Müller细胞吸光度值较正常糖浓度组明显降低(均P<0.01); 10、25、50、100μmol/L黄连素组细胞吸光度值较高糖浓度组明显升高(均P<0.05),且呈剂量依赖性; 5μmol/L黄连素组细胞吸光度值与高糖浓度组无明显差异(P>0.05)。

结论：黄连素在一定程度上能够减轻高糖对Müller细胞增殖活性的抑制作用,其作用强度与黄连素浓度呈正相关。     

视网膜脱离时神经生长因子对Müller细胞中间丝蛋白表达的影响

目的研究实验性视网膜脱离(RD)时外源性神经生长因子(NGF)对Müller细胞功能的影响作用.方法29只SD大鼠建立RD模型,并分为NGF实验组、空白、单纯RD、正常对照共4组.在建立RD模型后观察1.5、3、6h,1/2、1、2、4、8、16、32d.NGF实验组在建立模型后每4d在玻璃体腔内注射NGF 5μg/5μL/次,空白对照组注射空白载体作为对照.采用免疫组织化学方法对Müller细胞的GFAP和vimentin进行标记.非参数统计方法分析.结果单纯RD组显示随脱离时间延长,二者在Müller细胞的表达强度逐渐增强,分布范围变广.NGF实验组显示Müller细胞内的二者表达强度低于对照组,差异有显著性(P<0.05).结论外源性NGF能抑制RD后Müller细胞过度表达GFAP和vimentin,防止Müller细胞过度反应.     

Müller细胞在糖尿病视网膜病变中的研究进展

Müller细胞是脊椎动物视网膜最主要的神经胶质细胞,从内界膜到外界膜纵贯视网膜全层,参与构成血-视网膜屏障,积极参与视网膜发育并通过许多细胞内机制促进和维持视网膜稳态。Müller细胞在糖尿病视网膜病变的发生发展过程中扮演重要角色,其病理生理改变仍有待深入研究。本文就Müller细胞在糖尿病视网膜病变中的病理生理改变以及近年研究进展作一综述。     

阿柏西普对大鼠视网膜Müller细胞STAT3表达的影响

目的：探讨阿柏西普对大鼠视网膜Müller细胞信号转导及转录活化因子3(STAT3)表达的影响。

方法：采用不同浓度的阿柏西普 100μL(加药浓度分别为400、200、100pg/mL)大鼠永生化视网膜Müller细胞24、48h后,MTT法检测细胞增殖,流式细胞仪检测细胞凋亡,Transwell小室法检测细胞侵袭,Western-blot法检测AKT、STAT3蛋白表达。

结果：随着阿柏西普浓度的升高,Müller细胞的增殖活性下降(P<0.05)。处理48h后,随着阿柏西普浓度的升高,Müller细胞的凋亡率逐渐上升,Müller细胞的侵袭穿透指数逐渐降低(P<0.05)。转染48h后,随着阿柏西普浓度的升高,Müller细胞的AKT蛋白相对表达量无显著变化(P>0.05),STAT3蛋白相对表达量逐渐降低(P<0.05)。

结论：阿柏西普可抑制大鼠视网膜Müller细胞STAT3蛋白表达,从而抑制细胞增殖与侵袭性,促进细胞凋亡。     

阿柏西普对体外培养的视网膜Müller细胞膜离子通道的影响

目的：探讨阿柏西普对体外培养的高糖环境下视网膜Müller细胞膜K⁺通道的影响。

方法：人Müller细胞分为3组：对照组(常规DMEM培养基培养)、高糖组(高糖DMEM培养基培养)、实验组(高糖DMEM培养基和100μmol/L 阿柏西普处理)。采用荧光探针检测细胞K⁺浓度,MTT法检测细胞存活率,流式细胞仪检测细胞凋亡率,Western blot法检测Müller细胞caspase-3蛋白水平。

结果：Müller细胞培养48h后呈现谷氨酰胺合成酶(GS)阳性,纯化度在90%以上。荧光检测显示对照组、高糖组、实验组K⁺相对浓度分别为(2.14±0.44)%、(23.11±4.39)%、(5.20±0.92)%,细胞存活率分别为(100.00±0.00)%、(73.24±4.13)%、(85.22±5.33)%,细胞凋亡率分别为(5.03±1.91)%、(26.73±3.14)%、(16.63±2.73)%(均P<0.05)。 与对照组相比,高糖组Müller细胞内caspase-3蛋白水平显著上升(P<0.05); 与高糖组Müller细胞相比,实验组Müller细胞内caspase-3蛋白水平显著降低(P<0.05)。

结论：阿柏西普可抑制体外培养的高糖环境下视网膜Müller细胞膜K⁺通道,抑制高糖诱导的Müller细胞凋亡,降低caspase-3蛋白表达水平,促进细胞增殖。     

视网膜Müller细胞的研究现状

Müiler细胞是脊椎动物视网膜最重要的胶质细胞,了解其在健康视网膜的生理及功能和在病变视网膜的病理变化,可更好地理解在病变视网膜Müller细胞的胶质反应,探索有效的治疗措施.成年哺乳动物视网膜Müller细胞具有神经干细胞特性,在特定的条件下可能被激活,井受:Notch等一些信号通路的调控.视网膜Müller细胞在维持视网膜正常结构和功能中起重要作用,在病变视网膜中Müller细胞主要表现胶质增多反应,对神经元的功能既有保护又有损害,而Müller细胞的干细胞特性为视网膜神经元再生和对视网膜病变的治疗引出了新方向.     

视网膜Müller细胞的研究进展

Müller细胞是视网膜特化的胶质细胞,它贯穿于视网膜全层,与视网膜神经元、其它胶质细胞、视网膜血管等紧密联系。Müller细胞不但对视网膜正常发育起着决定性作用,而且能支持神经元活动、调节神经递质循环、维持细胞外环境平衡、调节视网膜血管通透性。视网膜Müller细胞代谢障碍将导致视功能丧失、神经元细胞死亡、视网膜水肿等。因此,Müller细胞对维持视网膜的正常生理功能起着重要作用。我们就近年来Müller细胞的研究进展作一综述。     

Nestin在视神经横断大鼠视网膜Muller细胞上的诱导表达

探讨中间丝蛋白nestin在视神经横断大鼠视网膜神经胶质细胞中的表达情况及可能的意义。 方法：制作大鼠视神经横断模型,分别于术后1,3,7d取材,制作视网膜矢状位冰冻切片,进行GS／nestin,免疫荧光双标。提取视网膜总RNA行nestin Real—time PCR半定量分析。 结果：正常大鼠视网膜中几乎看不到nestin阳性染色。术后1d,在Muller细胞上出现nestin的诱导表达,术后3d,nestin在Muller细胞上的表达进一步增强,术后1wk,nestin在Muller细胞上的表达保持在较高水平,Real-timePCR半定量分析与以上结果吻合。 结论：视神经横断后nestin在Muller细胞上的诱导表达是Muller细胞对视网膜损伤产生的一种反应,Nestin的表达量在一定时间内与病程进展相一致。Nestin在Muller细胞上的诱导表达尤其在足板处的强烈表达可能对视网膜神经节细胞具有保护作用。     

Blood-derived macrophages infiltrate the retina and activate Muller glial cells under experimental choroidal neovascularization

Inflammation is a major mechanism in the pathogenesis of age-related macular degeneration, the most important cause of blindness in the elderly. Previous studies have focused on the role of macrophages in regulating the growth of pathological new vessels over the retina, called choroidal neovascularization (CNV). However, no research has been done to evaluate the role of inflammation as a mechanism of vision loss and retinal degeneration in the retina underlying CNV. In other neuropathological conditions, hematogenous macrophages and/or resident microglia contribute to neurodegeneration. We have combined laser-induced CNV in mice and bone marrow transplantation with GFP-labeled bone marrow to determine the relative role of recruited blood-derived macrophages versus resident microglia in the retina associated with CNV. Using these chimeric mice, we have found that many GFP-labeled cells infiltrated the retina underlying CNV but not the retina unaffected by CNV. Immunostaining for the cell adhesion molecules VCAM 1, ICAM 1, and PECAM was strongly upregulated in retinal blood vessels under CNV. All GFP-labeled cells were immunoreactive for the macrophage marker F4/80. Most (70%) of the F4/80 immunoreactive cells were GFP-labeled under CNV. The density of resident microglia did not increase. Most GFP-labeled cells were found in close proximity to activated Muller cells. Depleting circulating macrophages with clodronic acid diminished the density of F4/80 immunoreactive cells as well as the density of pERK immunoreactive Muller cells in the retina under CNV. Thus, recruitment of blood-derived macrophages more than resident microglia seems to be associated with CNV.     

Functional diagnostics of retinal ischemia: Muller cells and neovascularization of the retina in diabetic retinopathy

Electrography was made for 58 patients (110 eyes) with proliferative diabetic retinopathy distinguished by neovascularization. Fluorescence angiography and electroretinography were dynamically made for 1-1.5 years in 18 patients with preproliferative diabetic retinopathy. The hyperfunction of Muller cells with an increasing glial index, which was detected earlier by us in progressing retinal ischemia, was also observed in retinal neovascularization. The regular monitoring of the dynamics of electroretinogram (ERG, made several times) makes it possible to evaluate in each separate case a degree of neovascularization risk. When the value of the glial index abruptly drops in a patient, who had earlier regular increases in the index value, there is a high probability of a fast development of retinal neovascularization. Regular ERG examinations are advisable for the evaluation of a neovascularization risk.     

Physiological properties of retinal Muller glial cells from the cynomolgus monkey, Macaca fascicularis--a comparison to human Muller cells

Retinae from rabbits and laboratory rodents are often used as 'models' of the human retina, although there are anatomical differences. To test whether monkey eyes provide a better model, a physiological study of Muller glial cells was performed comparing isolated cells and retinal wholemounts from the cynomolgus monkey, Macaca fascicularis and from man. The membrane conductance of Muller cells from both species was dominated by inward and outward K(+) currents. Cells displayed glutamate uptake currents and responded to nucleotides by intracellular Ca(2+) increases. However, there were also species differences, such as a lack of GABA(A) receptors and of Ca(2+)-dependent K(+) currents in monkey cells. Thus, the use of Muller cells from cynomolgus monkeys may be advantageous for investigating a few specific properties; in general, monkey cells are no more similar to human cells than those from standard laboratory animals.     

视网膜Müller细胞透明质酸酶功能分析

目的分析离体培养的视网膜Müller细胞对透明质酸的降解功能。方法提取的Mller细胞来自酶降解的猪后极部视网膜,并以3种不同细胞标记:胶质纤维酸性蛋白、波形蛋白、谷氨酰胺合成酶进行免疫荧光确认。细胞分为4组,其中1组培养液中加入透明质酸,2组培养液中加入透明质酸和抗integrinα2β1抗体,3组培养液中加入透明质酸和抗CD44抗体,4组培养液中未加入其他试剂。细胞预培养后,收集上清液。按配置聚丙烯酰胺凝胶的方法 ,加入透明质酸,不加入十二烷基硫酸钠制作底物胶。样品在底物胶电泳后,凝胶在酶降解缓冲液中培养24h,淋洗后以5g.L-1亮蓝染色、体积分数7%醋酸脱色,观察有无透明质酸降解后形成的脱色条带。结果 93.8%细胞胶质纤维酸性蛋白抗体染色阳性,94.6%细胞波形蛋白抗体染色阳性,88.6%细胞谷氨酰胺合成酶抗体染色阳性。培养的Mller细胞有降解透明质酸的功能,(4组)表现为蓝色背景(透明质酸染色)下的淡白色双条带(透明质酸降解后脱色);将细胞和透明质酸预培养可使脱色条带明显增厚,表现为明亮的白色条块(1组),同时加入透明质酸和integrinα2β1抗体产生相似的白色条块(2组),同时加入透明质酸和CD44抗体使脱色恢复为淡白色双条带(3组)。结论培养的Mller细胞有降解透明质酸的功能,细胞与透明质酸的相互作用明显加强细胞降解透明质酸的功能,CD44抗体可能减弱这种加强作用,integrinα2β1抗体无明显作用。     

视网膜Mueller细胞谷氨酸转运体及其功能调节

谷氨酸的兴奋毒性是造成视网膜神经节细胞损伤和死亡的主要因素之一,而谷氨酸转运体对维持细胞外谷氨酸水平起重要作用。GLAST是表达在Muller细胞膜上的一种重要的谷氨酸转运体,其在分子和蛋白质水平上的调节方式可能为酶底物—诱导的负反馈调节。     

Bipolar cells in the zebrafish retina

Zebrafish are an existing model for genetic and developmental studies due to their rapid external development and transparent embryos, which allow easy manipulation and observation of early developmental stages. The application of the zebrafish model to vision research has allowed for examination of retinal development and the characteristics of different retinal cell types, including bipolar cells. In particular, bipolar cell development, including differentiation, maturation, and gene expression, has been documented, as has physiological properties, such as voltage- and ligand-gated currents, and neurotransmitter receptor and ion channel expression. Mutant strains and transgenic lines have been used to document how bipolar cell connections and/or development may be altered, and toxicological studies examining how environmental factors may impact bipolar cell activity have been performed. The purpose of this paper was to review the existing literature on zebrafish bipolar cells, to provide a comprehensive overview of current information pertaining to this retinal cell type.     

视网膜中的Müller细胞

Muller细胞是视网膜的重要组成部分,除了传统认为其对视网膜神经细胞有支持和营养作用外,目前的研究表明Muller细胞在视网膜神经递质的调节、视网膜中钾离子的调节、视网膜中pH值的调节以及对神经细胞的信号传递方面都发挥了重要的作用.就Muller细胞的生理功能及其在视网膜病理状态下的改变作一综述.     

Interplexiform cells in macaque monkey retina

The presence of interplexiform cells in primate retina has been disputed, with the dopaminergic interplexiform cell in the New World monkey being the most fully understood. We have examined interplexiform cells in the Old World monkey using immunocytochemistry with the peroxidase-antiperoxidase method of visualization. In several species of macaque retina, two types of interplexiform cells are found. One stains with antisera to tyrosine hydroxylase, a biosynthetic enzyme for dopamine, and the other stains with antisera to gamma aminobutyric acid (GABA). The cell bodies of these two populations of interplexiform cells are located among the amacrine cells in the inner nuclear layer, and they send processes into both the inner and outer plexiform layers. GABA-positive interplexiform processes to the outer plexiform layer arise from the cell body while tyrosine hydroxylase-positive interplexiform processes most often originate from the heavily tyrosine hydroxylase-stained sublamina one of the inner plexiform layer. Cell-body diameter measurements and morphology suggest that these are different neuronal populations.