抗增生药物对人视网膜神经胶质细胞伯抑制作用

目的 寻找抑制视网膜、玻璃体内细胞增生的有效药物;明确抗增生药物秋水仙碱、道诺霉素和５－氟尿嘧啶（５－Ｆｕ）对体外培养的人视网胶质（ｒｅｔｉｎａｌ ｇｌｉａ,ＲＧ）细胞的作用。方法 用ＭＴＴ法测定秋水仙碱（０．５～１６．０ｕｇ／ｍｌ）、道诺霉素（０．１～３．２ｕｇ／ｍｌ）和５－Ｆｕ（０．５～１６．０ｕｇ／ｍｌ）对体外２人ＲＧ细胞的作用。结果 秋水仙碱（１．０～１６．０ｕｇ／ｍｌ）、道诺霉素（０．２     

人,狗,兔视网膜神经胶质细胞的原代培养

目的 了解同一方法能否培养不同哺乳动物的视网膜神经胶质细胞（ＲＧＣ）。方法 用酶消化法培养人,狗,兔的ＲＧＣ;根据细胞生长特点,电镜和免疫组织化学对照细胞进行鉴定。结果 培养细胞贴壁生长,形态不规则,生长周期较长（４周）电镜下可见细胞内均含有特征性中间丝（直径８～１０ｎｍ）免疫组化显示细胞呈神经胶质纤维酸性蛋白（ＧＦＡＰ）和波纹蛋白阳性,证实为ＲＧＣ,结论酶消化法是一种获取ＲＧＣ的可靠方法,用同一     

EGFR反义寡核苷酸对人视网膜神经胶质细胞增生的影响

目的 观察表皮生长因子受体（epidermal growth factor receptor,EGFR)反义寡核苷酸（antisense oligonucleotide,ASODN)对人视网膜神经胶质（retinal glial,RG)细胞增生的影响。方法 人视网膜神经胶质细胞的原代培养，用脂质体（Lipofectin)作为载体将修饰型EGFR ASODN转染RG细胞后，MTT法测细胞增生活性（吸光度A值）的变化。流式细胞仪观察细胞周期的变化。结果 EGFRASODN转染RG细胞后，细胞的增生活性降低，对RG细胞生长增生的抑制率约为46.5%，进入S期的细胞减少。结论 EGFRA-SODN能够抑制人RG细胞增生。     

人视网膜神经胶质细胞原代培养

采用组织消化培养法培养山人视网膜神经胶质(retinal glia,RG)细胞，用免疫组织化学技术对细胞进行鉴定，显示培养细胞呈神经胶质纤维酸性蛋白(glial fibrillary acid protein,GFAP)，S-100染色阳性。结合透射及扫描电镜观察，证实为人视网膜神经胶质细胞。 （中华眼底病杂志，1995，11：247-249）     

视网膜下液体对人视网膜神经胶质细胞生长的刺激作用与PVR关系

研究了２８例孔源性视网膜脱离患者视网膜下液（ＳｕｂｒｅｔｉｎａｌＦｌｕｉｄＳＲＦ）对人视网膜神经胶质细胞（ＲｅｔｉｎａｌＧｌｉａｌＲＧ）生长的刺激作用。结果表明：所有标本通常具有刺激ＲＧ细胞增殖能力，但存在较大范围的活动性，增殖率范围在基线上３６．４～１８１．８％，ＳＲＦ促ＲＧ细胞增殖能力与ＰＶＲ程度呈平行关系，ＰＶＲ在Ｃ级以上，促ＲＧ细胞增殖活力显著性加强（Ｐ＜０．０１）。     

低氧培养的人视网膜神经胶质细胞对血管内皮前体细胞的作用

目的探讨低氧环境下视网膜神经胶质细胞介导的低氧反应分子通路对血管内皮前体细胞(EPCs)的作用及其机制。方法人外周血循环EPCs(CD34+、CD33+双标阳性细胞)经荧光免疫激活流式细胞仪分选收集后培养;分别使用低氧(5%O2,实验组)和正常氧(20%O2,对照组)的人视网膜神经胶质细胞生长培养液作为条件培养液,多次处理EPCs,检测和分析EPCs的增殖活性(WST-1比色法)和分化能力(免疫荧光染色法);采用酶联免疫吸附试验检测实验组和对照组视网膜神经胶质细胞血管内皮生长因子(VEGF)的释放量,采用免疫印迹法检测细胞核内低氧反应因子(HIF)-1α的表达。结果实验组EPCs的增殖活性(吸光度值为1·53±0·10)和分化能力[细胞数目为(29±5)个]均较对照组增殖活性(吸光度值为0·88±0·25)和分化能力[细胞数目为(18±8)个]明显增强(P<0·05)。实验组培养6、24h后神经胶质细胞核内均可检测到HIF-1α表达,而对照组表达缺失。实验组视网膜神经胶质细胞VEGF的释放量较对照组明显增加,且具有时相性,培养24h实验组VEGF的释放量[(319·16±34·12)pg/ml]较对照组[(220·28±24·33)pg/ml]增加44·88%(P<0·01)。结论视网膜神经胶质细胞在低氧培养状态下可通过细胞因子的分泌作用直接促进培养的EPCs增殖和分化,这可能在EPCs参与的新生血管形成中发挥重要的介导作用,该作用与神经胶质细胞HIF-1α和VEGF的信号通道激活有关。本研究对神经胶质细胞介入血管前体细胞的病理生理作用进行了初步探讨。     

表皮生长因子受体反义寡核苷酸转染人视网膜神经胶质细胞

目的 观察表皮生长因子受体反义寡核苷酸经转染后，在胶质细胞中的通透性及稳定性表达。 方法 将异硫氰酸荧光素标记的硫代磷酸化修饰型及未修饰型的表皮生长因子受体反义寡核苷酸经阳离子脂质体介导或直接转染胶质细胞，分别于15、30 min、1 h后在荧光显微镜下观察胶质细胞中荧光分布情况。 结果 直接转染，修饰型及未修饰型表皮生长因子受体反义寡核苷酸30 min时少数胶质细胞胞浆可见点状荧光分布，4 h后，近50%的细胞可见荧光分布，修饰型可稳定存在于细胞中3~4 h，8 h后荧光消失；脂质体介导转染，表皮生长因子受体反义寡核苷酸15 min进入胶质细胞，4 h后70%～80%的胶质细胞可见荧光分布，非修饰型4 h荧光消失，修饰型可稳定存在于细胞中10～12 h，14 h后荧光消失。 结论 脂质体包裹的修饰型表皮生长因子受体反义寡核苷酸能进入胶质细胞内并稳定表达。 （中华眼底病杂志，2003，19：52-54）     

在人羊膜上培养牛视网膜神经胶质细胞的初步探讨

目的　研究在具有完整基底膜的人羊膜上培养牛视网膜神经胶质 (RG)细胞的可能性。方法　用常规培养液进行原代及传代牛RG细胞的培养 ,并以GFAP及S 10 0染色进行免疫组化鉴定。取第 3代或第 4代牛RG细胞 ,以 1 0× 10 5/mL密度接种于羊膜基底膜面进行培养 ,并进行上述同样的鉴定。结果　 ( 1)原代RG细胞GFAP及S 10 0染色阳性细胞分别为 86 2 %和 83 5 % ,传代RG至第 3代时GFAP及S 10 0染色阳性细胞百分数明显增加 ,分别为 92 8% ,90 3 %。 ( 2 )培养的牛RG细胞的羊膜基底膜面大部分细胞阳性着色 ,其GFAP和S 10 0染色阳性细胞分别为 91 4%和90 1%。结论　牛RG细胞可以在人羊膜基底膜面生长。     

EGFR反义寡核苷酸对人视网膜神经胶质细胞迁移的影响

目的:观察表皮生长因子受体(epidermal growth factor receptor,EGFR)反义寡核苷酸(antisense oligonucleotide,ASODN)对人视网膜神经胶质(retinal glial,RG)细胞迁移的影响。方法:人视网膜神经胶质细胞的原代培养,用脂质体(Li-pofectin)作为载体将修饰型EGFR ASODN转染RG细胞后,采用Murphy细胞计数方法评价反义寡核苷酸对人RPE细胞体外迁移的影响。结果:脂质体介导EGFR反义寡核苷酸组与对照组比较,细胞迁移活性受到明显抑制(P<0.05),转染24h后抑制率63.27%。结论:EGFR ASODN能够抑制人RG细胞迁移。     

Cytotoxic effects of antiproliferative agents on human retinal glial cells in vitro

Purpose: Proliferative vitreoretinopathy (PVR) is characterizedby the formation of cellular membranes on the detached retina and also in the vitreous. Glial cellscan be found in epiretinal and subretinal membranes from eyes with PVR, proliferative diabeticretinopathy (PDR), idiopathic macular pucker, uveitis and other diseases affecting theretina. Proliferation and contraction of glial cells appears to play a role in the pathogenesisof PVR. This study is designed to inspect the effectiveness of harringtonine, as well as colchicine,daunomycin and fluorouracil, against cellular proliferation of cultured human retinal glial cellsthat might be involved in the retinal and/or vitreous proliferation. Methods: Cultures of human retinal glial cells were preparedusing the enzyme digesting method. Cells that had been in culture for 2–5 passages were usedin this study. Harringtonine (0.063 g/ml 2.0 g/ml), colchicines(0.5 g/ml 16.0 g/ml), daunomycin (0.1 g/ml 3.2 g/ml) and 5-fluorouracil(0.5 g/ml 16.0 g/ml) were added to cultures of human retinal glial cellsand the proliferation rates of the cells were measured by the MTT method. Results: Harringtonine at the dosage of 0.063 g/mlinduced suppression of cellular growth, but the changes were not statistically significant (p > 0.05).At a dosage ranging from 0.125 g/ml to 2.0 g/ml, harringtonine significantly suppressedcellular growth according to the test (p < 0.01). Likewise, other antiproliferativeagents inhibited cellular growth significantly at a dosage from 1.0 g/ml to 16.0 g/ml(colchicine), 0.2 g/ml to 3.2 g/ml (daunomycin) and 1.0 g/ml to 16.0 g/ml(5-fluorouracil), but not at 0.5 g/ml (colchicine), 0.1 g/ml (daunomycin) and0.5 g/ml (5-fluorouracil). The ID₅₀ were 0.33 g/ml (harringtonine), 3.11 g/ml (colchicine), 0.79 g/ml (daunomycin) and 5.23 g/ml (5-fluorouracil), respectively.Conclusions: Harringtonine was extremely effective ininhibiting human retinal glial cell proliferation, like other antiproliferative drugs such as colchicine,daunomycin and 5-fluorouracil. Harringtonine, therefore, may be a candidate for further studies regardingthe treatment of experimental PVR.     

吲哚青绿对人视网膜色素上皮细胞和神经胶质细胞的影响

目的 :研究吲哚青绿 (indocyaninegreen ,ICG)溶液对体外培养的人视网膜色素上皮细胞 (retinalpigmentepitheli um ,RPE)和视网膜神经胶质细胞 (retinalglialcells ,RGC)的毒性。方法 :将ICG分别溶解于注射用水、乳酸林格氏缓冲液和无血清培养基 ,配制成 0 2 5、0 0 5、0 0 2 5mg/ml浓度的ICG溶液。测定各种溶液的渗透压与pH值。采用以上三种溶液作用于第 3代人RGC和RPE细胞 ,1、5、10分钟后更换为含 10 %小牛血清的DMEM培养基 ,37℃继续孵育 2 4小时。检测细胞形态和细胞生存率。结果 :本实验浓度的乳酸林格氏液组与无血清培养基组的ICG未引起RPE与RGC细胞形态改变 ,但随浓度和时间增加 ,两种细胞吸光度值降低 (P <0 0 5 )。低浓度注射用水组ICG溶液明显抑制细胞生存 (P <0 0 1)。结论 :ICG对人RPE和RGC细胞有一定毒性。ICG溶液对RPE和RGC细胞生长的抑制具有浓度和时间依赖性。对两种细胞活性的抑制无选择性。使用低浓度ICG ,溶解于等渗溶剂 ,缩短ICG接触时间 ,可减轻其对视网膜细胞的毒性作用。     

A comparative study of effects of antiproliferative drugs on human retinal pigment epithelial cells in vitro.

The effects of various antiproliferative drugs have been tested in numerous cell types in vitro, but a comparison of effects of these drugs on cultured human retinal pigment epithelium (RPE) has not been reported. We studied the effects of four most widely used antiproliferative drugs (5-FU, daunomycin, mitomycin and dexamethasone) in a new in vitro model system (cultured human RPE). Various concentrations and exposure periods were tested in four human RPE cell lines. 5-FU showed a dose-dependant growth inhibition, with an ID50 of 5.35 +/- 2.38 x 10(-7) M after 4 days culture. A decrease of cell number occurred relatively later, the slope of the dose-response curve was less steep than that of others. Mitomycin C showed an immediate and strong growth inhibition and cytotoxic effects, with an ID50 of 3.73 +/- 0.71 X 10(-9) M. RPE cultured with daunomycin showed an abrupt decrease of cell number from 10(-9) M to 10(-8) M, the ID50 value was 1.07 +/- 0.23 x 10(-8) M. Dexamethasone showed a biphasic effect; it stimulated cell growth at 10(-7) to 10-6 M and inhibited cell growth at 10(-4) M or higher, with an ID50 of 6.05 +/- 1.61 x 10(-3) M. The advantages and disadvantages of these drugs and the prospective clinical application of these drugs for management of proliferative vitreoretinopathy (PVR) were discussed. Development of an in vitro model using cultured human RPE to study the effects of various antiproliferative drugs can provide a rapid, safe and inexpensive method for selection of drugs used for management of proliferative vitreoretinopathy.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.     

褪黑素对体外培养的人视网膜色素上皮细胞氧化损伤的保护作用

Objective To investigate the protective effects of melatonin on the retinal pigment epithelium (RPE) against oxidative damage induced by hydrogen dioxide (H2O2) and its mechanism. Methods The RPE cells were seeded and divided into normol control group, oxidative damage group and the treatment group (treated with melatonin at the concentration of 1×10.7 mol/L,1×10-6 mol/L, 1×10-5 mol/L and 1×10-4 mol/L). The model of oxidative damage on the RPE cells was established by culturing the RPE cells with H2O2 at the concentration of 600 μmol/L for 1 hour in vitro. The cell viability of RPE cells was detected by the methyl thiazolyl tetrazolium (MTT) method. The degree of oxidative damage was evaluated by detecting the superoxide dismutase (SOD) and maleic dialdehyde (MDA). Apoptosis was detected qualitatively using the DNA Ladders electrophoretic mothed, and quantitatively using the Annexin V-FITC/PI double staining flow cytometry. Results Compared with normal control group, the oxidative damage group had low cell viability, low SOD and high MDA contents, and high apoptosis rate(t=2.25,39.50,68.42;P<0.05). Compared with oxidative damage group, the treatment group had high cell viability, high SOD and low M DA contents, and low apoptosis rate (P<0.05). Conclusions Melatonin has a protective effect on the RPE against oxidative damage induced by H2O2. The mechanism may involve in reinforcing the cell viability, strengthening the activity of antioxidase, and reducing the apoptosis.