兔视网膜挫伤后Müller细胞Vimentin表达的变化

目的:探讨兔视网膜挫伤后Müller细胞波形蛋白(Vimentin)表达的变化方法:通过3J能量自由落体方式制作兔眼挫伤性视网膜病变模型,于伤后1/8,1,3,7,14d时处死动物取材,免疫组化染色和计算机图像分析仪检测视网膜挫伤后Müller细胞Vimentin的表达和分布.结果:视网膜挫伤后1d Vimentin开始阳性表达增强,7d达到高峰,14d略有下降.随着视网膜挫伤时间的延长,Vimentin的免疫染色范围也逐渐向外扩展,3d时免疫染色达外界膜,7d时视网膜全层都有表达,二组比较,各时段差别均P＜0.01,存在统计学差异.结论:视网膜挫伤后Müller细胞Vimentin反应动态增强.     

bFGF对兔视网膜Müller细胞表达VEGF的影响

血管内皮生长因子（VEGF）和碱性成纤维细胞生长因子（bFGF）是视网膜细胞分泌的两种最重要的血管生成因子。二者有协同作用，bFGF还可以诱导内皮细胞表达VEGF。视网膜Müller细胞是视网膜中分泌VEGF的主要细胞之一，为了进一步探讨Müller细胞在糖尿病视网膜病变（DR）中的作用，本研究观察了bFGF对体外培养免视网膜Müller细胞表达VEGF的影响。     

金纳多对高糖所致视网膜Müller细胞合成谷胱甘肽减少的保护作用

目的研究高浓度葡萄糖对体外培养兔视网膜Müller细胞合成谷胱甘肽（GSH）的影响及自由基清除剂金纳多（EGb761）的作用。方法采用比色法测定不同浓度葡萄糖（5.5、30、40、50mmol/L）下,兔Müller细胞合成GSH的质量分数。在50mmol/L葡萄糖培养液中加入不同质量浓度EGb761（0、5、10、15、20、25、30、45、90μg/mL）培养3d后测定兔视网膜Müller合成GSH的质量分数。结果各浓度葡萄糖均使兔视网膜Müller细胞合成GSH的质量分数减少。加入各质量浓度EGb761后GSH合成增加,质量浓度为10μg/mL时增加最高。结论EGb761对高浓度葡萄糖致兔视网膜Müller细胞合成GSH质量分数减少起保护作用。     

豚鼠近视眼视网膜Müller细胞的原代培养

目的研究豚鼠近视眼视网膜Müller细胞原代培养的方法。方法用半透明眼罩遮盖法建立豚鼠的近视眼模型。采用酶消化法培养豚鼠近视眼视网膜Müller细胞,用倒置相差显微镜观察Müller细胞的生长状况,以GFAP、Vimentin免疫组化染色进行细胞鉴定。结果半透明眼罩遮盖豚鼠右眼2周后,遮盖眼眼轴延长,近视形成。原代培养的视网膜Müller细胞贴壁生长,胞体较大、扁平,GFAP、Vimentin免疫组化染色呈阳性。结论酶消化法是培养豚鼠近视眼视网膜Müller细胞的一种有效方法。     

体外培养视网膜Müller细胞高压气体冲击损伤模型

目的建立一种视网膜Müller细胞原发性创伤性损伤模型，以探讨视网膜Müller细胞在视网膜损伤中的作用。方法取体外培养的SD大鼠视网膜Müller细胞，随机分为对照组和致伤组，待细胞融合后与自行研制的新型高压气体冲击细胞装置相连接，致伤组通过计算机设置25、50、100、150、200、250kPa不同的驱动压力引发直接冲击力作用Müller细胞。致伤后观察细胞形态，台盼蓝染色检测细胞活性，培养上清检测乳酸脱氢酶（LDH）浓度的改变。结果致伤后形态学显示明显改变，细胞肿胀，线粒体变形。LDH释放率测定显示25kPa驱动压力下的直接冲击力即可引起细胞膜通透性改变，200kPa时细胞已达到最大程度的损伤，差异有统计学意义（P〈0．05）。结论视网膜Müller细胞的高压气体冲击损伤模型是一种新型的在细胞水平研究创伤性损伤的原发性损伤模型。     

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。     

Müller细胞对大鼠光感受器变性及视功能的保护作用

目的以RCS视网膜色素变性大鼠为模型,研究经诱导产生神经干细胞特性的Müller细胞视网膜下腔移植对光感受器变性的保护作用。方法向6周龄VC大鼠玻璃体腔内注射神经毒素混合液（包含NMDA、kainate、FGF2和胰岛素）刺激视网膜Müller细胞。1周后取材分离Müller细胞体外原代培养。将细胞标记后,以每眼1×10^5个细胞密度移植到6周龄RCS大鼠右眼视网膜下腔,左眼分组注射正常Müller细胞和PBS作为同型对照及阴性对照。术后分别于第1、3、5、7周,行视网膜铺片、组织病理学及视觉电生理检查。结果培养的Müller细胞纯度可达96％以上,其中表达神经干细胞标志物的细胞占总细胞量的53％以上。组织病理学观察显示,在相同时间点移植眼保留的光感受器细胞数量明显较同型对照眼多,同型对照移植眼保留的光感受器细胞数量明显较阴性对照眼多。视网膜电图（ERG）检查结果与病理学结果相符。结论视网膜下腔移植经诱导产生神经干细胞特性的Müller细胞可以有效延缓RCS大鼠视网膜光感受器变性,为治疗视网膜变性疾病提供了新的途径。     

豚鼠形觉剥夺性近视眼视网膜Müller细胞中bFGF的表达

目的观察视网膜Müller细胞中碱性成纤维细胞生长因子（bFGF）在豚鼠形觉剥夺性近视（FDM）中的表达,进一步探讨近视眼的发病机制。方法用半透明眼罩遮盖法建立豚鼠FDM模型;采用玻璃体腔注射L-α-氨基己二酸（L-α-AAA）破坏视网膜Müller细胞,观察其对豚鼠FDM形成的影响。视网膜检影验光测屈光度,A型超声测量眼轴长度。免疫组织化学法观察bFGF在视网膜Müller细胞中的表达。结果遮盖眼与对照眼相比,产生了相对性近视（P〈0.05）,眼轴增长（P〈0.05）;去遮盖后,近视度数降低（P〈0.05）;破坏视网膜Müller细胞后,近视度数较单纯遮盖眼降低（P〈0.05）。近视眼与对照眼相比,视网膜Müller细胞中bFGF表达减弱。结论视网膜Müller细胞可能参与了FDM形成的调控;视网膜Müller细胞中bFGF的表达可能与FDM有关。     

糖尿病视网膜Müller细胞GLAST功能变化的研究进展

糖尿病引起的视网膜神经功能异常可能先于血管损伤,引起这一改变的最主要原因是视网膜细胞外谷氨酸的大量蓄积,持久激活谷氨酸受体以及损伤视网膜神经元。细胞外谷氨酸的蓄积可能与视网膜Müller细胞L-谷氨酸/L-天冬氨酸转运体(GLAST)功能下降有关,致使细胞外的谷氨酸不能及时转运到Müller细胞内。本文就Müller细胞GLAST的作用机制及在糖尿病状态下功能的变化进行综述。     

糖尿病对视网膜Müller细胞功能的影响

视网膜M櫣ller细胞在结构上与视网膜神经元、血管关系密切,其主要功能是调节细胞外间质中的离子浓度、参与谷氨酸代谢、调节视网膜内酸碱平衡、支持视网膜内各种细胞代谢等。在糖尿病视网膜病变中,视网膜M櫣ller细胞功能的损害可引起K 离子通道功能的损害,影响视网膜的血管功能;谷氨酸载体活性的损害和谷氨酰胺合成酶的功能降低,可影响神经元细胞的活性和功能;改变GFAP和occludin的表达与分布,使血-视网膜屏障受到损害;向成纤维细胞转化产生收缩力,参与增生性糖尿病视网膜病变的发生与发展;碳酸酐酶功能受到影响,引起视网膜内酸碱平衡失调。     

bFGF对兔眼视网膜的影响挫伤Muller细胞Vimentin表达

目的研究碱性成纤维细胞生长因子（bFGF）对兔视网膜挫伤后Mtiller细胞的影响。方法26只白兔通过3J能量自由落体的方式制作兔眼挫伤性视网膜病变模型,随机分为bFGF实验组、生理盐水对照组、单纯挫伤组、正常对照组。其中正常对照组2只（4只眼）,单纯挫伤组4只（8只眼）,bFGF实验组和生理盐水对照组各10只（20只眼）。bFGF实验组每2d玻璃体腔内注射bFGF2μg（10μL）,生理盐水对照组注射生理盐水10μL。采用免疫组织化学染色检测视网膜挫伤后3h,1、3、7、14d各时间点Miiller细胞波形蛋白（Vimentin）的表达。结果bFGF实验组视网膜Muller细胞Vimentin的表达高于生理盐水对照组,且呈上升趋势,两组比较各时段差异均有统计学意义（P〈0．05）。结论bFGF可以增强视网膜Muller细胞Vimentin的阳性表达,加速Muller细胞的活化,促进损伤修复。     

雌激素对缺氧视网膜Müller细胞色素上皮衍生因子表达的影响

目的:探讨雌激素对缺氧视网膜Müller细胞色素上皮衍生因子（P EDF）表达的影 响。 方法:采用逆转录聚合酶链反应（RT PCR）和Western blotting印 迹分析方法分别测定不同浓度（10^-7、10^-6、10^-5 mmol/L）雌二醇 （E₂）作用于缺氧Müller细胞后，细 胞内 PEDF mRNA及蛋白表达水平。 结果:缺氧24 h后PEDF mRNA及蛋白表达明 显降低， 10^-7、10^-6、10^-5 mmol/L E₂作用于Müller细胞后可明显缓解 由于缺氧引起的细胞内PEDF mRNA及蛋白表达的降低，并与E₂的浓度有关。 结论:雌激素可以调控PEDF的表达 ，其可能在雌激素对视网膜新生血管形成的调控中起重要作用。     

Genetic deletion of COX-2 diminishes VEGF production in mouse retinal Müller cells

Non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit COX activity, reduce the production of retinal VEGF and neovascularization in relevant models of ocular disease. We hypothesized that COX-2 mediates VEGF production in retinal Müller cells, one of its primary sources in retinal neovascular disease. The purpose of this study was to determine the role of COX-2 and its products in VEGF expression and secretion. These studies have more clearly defined the role of COX-2 and COX-2-derived prostanoids in retinal angiogenesis. Müller cells derived from wild-type and COX-2 null mice were exposed to hypoxia for 0-24 h. COX-2 protein and activity were assessed by western blot analysis and GC-MS, respectively. VEGF production was assessed by ELISA. Wild-type mouse Müller cells were treated with vehicle (0.1% DMSO), 10 μM PGE₂, or PGE₂ + 5 μM H-89 (a PKA inhibitor), for 12 h. VEGF production was assessed by ELISA. Hypoxia significantly increased COX-2 protein (p < 0.05) and activity (p < 0.05), and VEGF production (p < 0.0003). COX-2 null Müller cells produced significantly less VEGF in response to hypoxia (p < 0.05). Of the prostanoids, PGE₂ was significantly increased by hypoxia (p < 0.02). Exogenous PGE₂ significantly increased VEGF production by Müller cells (p < 0.0039), and this effect was inhibited by H-89 (p < 0.055). These data demonstrate that hypoxia induces COX-2, prostanoid production, and VEGF synthesis in Müller cells, and that VEGF production is at least partially COX-2-dependent. Our study suggests that PGE₂, signaling through the EP₂ and/or EP₄ receptor and PKA, mediates the VEGF response of Müller cells.     

Expression of Cre recombinase in retinal Müller cells

Purpose

In an effort to generate inducible RPE-specific Cre mice using a 3.0-kb human vitelliform macular dystrophy-2 (VMD2) promoter, we identified a mouse line with unanticipated Cre activity in the neural retina, including Müller glial cells. Müller cells play important roles in the function and maintenance of the retina, and this mouse line would be potentially useful for conditional gene targeting in Müller glia. We therefore characterized the timing, inducibility, and cell specificity of Cre expression, as well as Müller cell-specific efficiency of Cre-mediated recombination in this mouse line.

Methods

Transgenic mice carrying cassettes of human P_VMD2-rtTA and TRE-cre were generated. Cre expression was characterized using a Cre-activatable lacZ reporter mouse line (R26R) and a floxed interleukin six signal transducing receptor (gp130) mouse line.

Results

β-Galactosidase (β-gal) assay and immunohistochemical analysis of VMD2-cre/R26R double transgenic mice indicated that Cre activity was detected in cells located in the inner nuclear layer, with prominent expression of β-gal in Müller cells. Cre activity was also detected in photoreceptors in the outer nuclear layer. PCR analysis demonstrated that Cre-mediated recombination initiated by embryonic day 15. Immunohistochemical analysis indicated that Cre-mediated deletion of floxed gp130 gene occurred in 52% of the retinal Müller cells. Retinal function and morphology were normal in 10-month-old VMD2-cre mice.

Conclusion

We generated a transgenic cre mouse that is useful to study gene activation and inactivation in retinal Müller cells.     

Regulation of brain-derived neurotrophic factor on the expression of GLAST protein and its function in retinal Müller cells of mice

Objective To study the effect of brain-derived neurotrophic factor (BDNF)on the expression of L-glutamate/L-aspartate transporter (GLAST) protein and its function in the retinal mice at 3 to 7 days postnatal were cultured by an enzymatic digestion method, and the third passage different concentrations of recombinant human BDNF (50, 75, 100, 125 and 150 ng/ml) for 24 h in group and the control group. The expression of GLAST protein was analyzed with one-way analysis of variance (ANOVA) and L-[3,4-3H]-glutamic acid uptake was analyzed with independent samples t test. Results The expression of GLAST protein in the control group was 0.151±0.025 and the expression in the BDNF group (50, 75, 100, 125 and 150 ng/ml) was 0.331±0.076, 0.413±0.110, 0.497±0.080, 0.411±0.072, and 0.319±0.084, respectively. Different concentrations of BDNF could up-regulate the expression of GLAST protein compared to the control group (F=6.793, P=0.003).The expression of GLAST protein reached a maximum when the concentration of BDNF was 100 ng/ml.L-[3,4-3H]-glutamic acid uptake for the 100 ng/ml BDNF group and control group was 81 213±significantly higher than for the control group (t=6.462, P=0.023). Conclusion BDNF can up-regulate the expression of GLAST protein and increase extracellular glutamate uptake.     

Primary culture of retinal Müller cells in adult rats assisted by the peritoneal exudative cells

Objective To investigate the effect of peritoneal exudative cells as feeder cells on growth from adult rats, which were identified with specifically biological marker of maerophage (CD68). The by enzyme digestion method, and identified by GFAP and vimentin immunocytochemicaUy. As the feeder cells. Results Over ninety-five percent of rat peritoneal exudative cells were macrophage, which have a flask and grew fast, with large applanate cell bodies. The third-generation cells grew slowly. After cocuhured with feeder cells, the Moiler cells showed more rapid growth rate with more cells in S and G2/M phase(S phase, t=4. 172, P<0.001; G2/M phase, t=3. 562, P<0.01)and less apoptotic rate (t =3. 804, P<0. 01). The growing cycle was cut down from 25-30 days to 18-22 days for the first-generation cells, from 10-15 days to 7-10 days for the second-generation cells. Conclusion It is an effective method to use the peritoneal exudative cells as feeder cells cocuhured with primary culture of retinal Müller cells,which can shorten the culture period of Müller cells in adult rats.     

Primary culture of retinal Müller cells in adult rats assisted by the peritoneal exudative cells

Objective To investigate the effect of peritoneal exudative cells as feeder cells on growth from adult rats, which were identified with specifically biological marker of maerophage (CD68). The by enzyme digestion method, and identified by GFAP and vimentin immunocytochemicaUy. As the feeder cells. Results Over ninety-five percent of rat peritoneal exudative cells were macrophage, which have a flask and grew fast, with large applanate cell bodies. The third-generation cells grew slowly. After cocuhured with feeder cells, the Moiler cells showed more rapid growth rate with more cells in S and G2/M phase(S phase, t=4. 172, P<0.001; G2/M phase, t=3. 562, P<0.01)and less apoptotic rate (t =3. 804, P<0. 01). The growing cycle was cut down from 25-30 days to 18-22 days for the first-generation cells, from 10-15 days to 7-10 days for the second-generation cells. Conclusion It is an effective method to use the peritoneal exudative cells as feeder cells cocuhured with primary culture of retinal Müller cells,which can shorten the culture period of Müller cells in adult rats.     

Primary culture of retinal Müller cells in adult rats assisted by the peritoneal exudative cells

Objective To investigate the effect of peritoneal exudative cells as feeder cells on growth from adult rats, which were identified with specifically biological marker of maerophage (CD68). The by enzyme digestion method, and identified by GFAP and vimentin immunocytochemicaUy. As the feeder cells. Results Over ninety-five percent of rat peritoneal exudative cells were macrophage, which have a flask and grew fast, with large applanate cell bodies. The third-generation cells grew slowly. After cocuhured with feeder cells, the Moiler cells showed more rapid growth rate with more cells in S and G2/M phase(S phase, t=4. 172, P<0.001; G2/M phase, t=3. 562, P<0.01)and less apoptotic rate (t =3. 804, P<0. 01). The growing cycle was cut down from 25-30 days to 18-22 days for the first-generation cells, from 10-15 days to 7-10 days for the second-generation cells. Conclusion It is an effective method to use the peritoneal exudative cells as feeder cells cocuhured with primary culture of retinal Müller cells,which can shorten the culture period of Müller cells in adult rats.