The Effect of Zinc on the Apoptosis of Cultured Human Retinal Pigment Epithelial Cells

To clarify the effects of zinc on the proliferation and apoptosis of cultured human retinal pigment epithelia (RPE) and the expression of caspase 3 in RPE cells. The effect of Zinc on theproliferation of RPE were examined with MTT method. TUNEL method was used to detect the apoptosis of RPE cells. Caspase 3 was detected by immunohistochemistry. A concentration of zinc higher than 0.001 μM could inhibit the proliferation of RPE. And the relationship between concentration of zinc higher than 10 μM and growth prohibition rate of RPE cells was dose dependent. All concentrations of zinc including 0.001 μM enhanced the expression of caspase 3 of RPE. But only the concentration of zinc higher than 0.01 μM could induce apoptosis of RPE. It is concluded that zinc could enhance the expression of caspase 3 of RPE cells and induce apoptosis of RPE cells. Caution should be taken when using zinc supplements for the treatment of ARMD patients without deficiency of zinc.

Lysosomal Enzyme Activities in Cultured Retinal Pigment Epithelial and Glial Cells of RCS Rat

Purpose: To compare the activities of acid phosphatase, N-acetyl-β-glu-cosaminidase and a- mannosidase in cultured retinal pigment epithelium (RPE)and glial cells of Royal College of Surgeons (RCS) rat with those in Long Evans(LE).Methods: The cultured RPE and glial cells of RCS and LE rat were plated into thesame 96 well microtitre, and the biochemical method in microsystem were usedfor enzyme assays.Results: The activities of acid phosphatase and N-acetyl- β-glucosaminidase arehigher by, respectively, 30% and 46% in cultured RPE of RCS rat than LE rat.The activity of a- mannosidase has no significant difference. The activities of 3enzymes in the retinal glial cells derived from RCS rats are higher than LE rat by43% to 77%.Conclusion: These results suggest that the high activities of lysosomal enzymes inRCS RPE and glial cells may play an important role in the pathogenesis of retinaldystrophy. Eye Science 1996; 12:20-27.

Modification of Isolation and Culture of Human Retinal Pigment Epithelial Cells

Purpose:To modify the isolation of human retinal pigment pithelial(RPE)cells and to increase the purification and production of cultured RPE cells.Methods:The human eyecups were fixed on a fubber holder.After digestion by trypsin,RPE cells were collected,then cultured and identified by morphology,immunohistochemistry and electron microscopy.Results:The cultured RPE cells grew actively in the early stage with transparent nucleus and abundant melanin particles in cytoplasm.These cells were positive in DOPA oxidase reaction and in anti-pancytokeratin antibody staining.Cellular microvilli and tight junctions could be seen through transmission electrom microscopy.Conclusion:We developed a rubber holder to fix the eyecup.Using this holder,more and purer cultured RPE cells can be obtained.These cultured REP cells are similar to those in vivo in morphology and immunohistochemical staining.

New antioxidant SkQ1 is an effective protector of rat eye retinal pigment epithelium and choroid under conditions of long-term organotypic cultivation

Cells have intrinsic mechanisms for cleaning harmful oxidants represented mainly by reactive oxygen species (ROS). Despite the antioxidant defense, ROS can cause serious damage to the retina that with age leads to various eye diseases and even blindness. Among numerous cell sites of ROS generation, mitochondrial electron transport is of crucial importance. Recently, for the purpose of cleaning ROS in the mitochondrial matrix, powerful mitochondria- targeted antioxidant “SkQ1” has been invented. We studied SkQ1 effects upon tissues of rat posterior eye cup that consisted: retinal pigment epithelium (RPE) ? choroidal coat ? scleral coat. The eye cups were isolated from the eyes of adult albino rats and cultivated in rotary tissue culture system in the presence of 20 nM SkQ1 or without this compound. After 7 days - 1 month in vitro eye cup samples were studied by immunohistochemistry, routine histology, morphometry, and digital image analysis. We have found that under chosen, “in vitro like in vivo” conditions 20 nM SkQ1 effectively reduced cell death in RPE and choroid, protected RPE from disintegration caused by cell phenotypic transformation and withdrawal from the layer, suppressed transmigration of choroidal coat cells. In the ex vivo model we used degenerative processes were more pronounced in the eye cup center where SkQ1 effect was most vivid. All this give us hopes for effectiveness of SkQ1 treatment of retinal central part that is very susceptible to light-induced over-oxidation injury and mostly suffering in many age-related diseases, AMD, in particular.

Shaping the eye from embryonic stem cells: Biological and medical implications

Organogenesis is regulated by a complex network of intrinsic cues, diffusible signals and cell/cell or cell/matrix interactions that drive the cells of a prospective organ to differentiate and collectively organize in three dimensions. Generating organs in vitro from embryonic stem (ES) cells may provide a simplified system to decipher how these processes are orchestrated in time and space within particular and between neighboring tissues. Recently, this field of stem cell research has also gained considerable interest for its potential applications in regenerative medicine. Among human pathologies for which stem cell-based therapy is foreseen as a promising therapeutic strategy are many retinal degenerative diseases, like retinitis pigmentosa and age-related macular degeneration. Over the last decade, progress has been made in producing ES-derived retinal cells in vitro, but engineering entire synthetic retinas was considered beyond reach. Recently however, major breakthroughs have been achieved with pioneer works describing the extraordinary self-organization of murine and human ES cells into a three dimensional structure highly resembling a retina. ES-derived retinal cells indeed assemble to form a cohesive neuroepithelial sheet that is endowed with the intrinsic capacity to recapitulate, outside an embryonic environment, the main steps of retinal morphogenesis as observed in vivo. This represents a tremendous advance that should help resolving fundamental questions related to retinogenesis. Here, we will discuss these studies, and the potential applications of such stem cell-based systems for regenerative medicine.

Influence of Cell Confluency on the Expression of the α4 Integrin Subunit of Retinal Pigment Epithelial Cells

Integrins are a family of transmembrane glycoproteins that mediate cell-cell and cell-extracellular matrix interactions. The integrin α4 subunit is widely expressed by cells from the immune system and its expression by non-hematopoietic cells is scarce. In the present study, gene and protein expression of this integrin subunit was characterized in proliferating and quiescent human RPE cells. Immunofluorescent studies confirm that the α4 subunit is expressed in vitro by RPE cells, a result that has been validated by immunofluorescence and FACS analyses. The accumulation of the α4 integrin at cell-cell junctions in post-confluent RPE cell cultures negatively correlated with the level of expression of the mRNA transcript. Accordingly, transient transfection analyses reveal that the α4 promoter activity is considerably reduced when RPE cells form a confluent monolayer. Moreover, transfection of recombinant constructs bearing 5’-deletions of the α4 promoter segment allows the localization of strong negative regulatory elements on the -76 to -300 region of the α4 gene suggesting that its expression is intimately linked to the proliferative state of primary cultured RPE cells.

TIMP-1 Production in Human Retinal Pigment Epithelial Cells after Laser Exposure

Purpose: To investigate changes in the production of tissue inhibitor of metalloproteinase type 1 (TIMP-1) by human retinal pigment epithelial (RPE) cells following argon laser exposure.Methods: Human cultured ARPE19 cells were exposed to argon green laser at four different energy levels ranging from 60mW to 360mW. After laser exposure, the culture media were sampled at 0, 24, 72 and 144 hours for TIMP-1 concentration produced by the RPE cells. The levels of TIMP-1 in the cells treated with different laser energy levels were compared with a control group not exposed to laser application.Immunocytochemistry for proliferating cell nuclear antigen (PCNA) was performed to detect any adverse effects on the RPE cells caused by laser exposure.Results: Immediately after laser exposure, the concentration of TIMP- 1 was not detectable. At 24 hours after laser exposure, the concentration of TIMP-1 increased significantly in RPE cells treated with 120mW and 240mW at 24 hours (P=0.006 and P=0.001respectively) compared with control cells. At 72 hours after treatment, RPE cells treated at 120mW, 240mW and 360mW demonstrated significantly increase in TIMP-1production compared with control (P=0.003, P ＜ 0.001 and P ＜ 0.001, respectively).No significant reduction in cell viability was observed following laser application as detected by PCNA expression.Conclusions: Our results demonstrated that early TIMP-1 production by RPE cells in cell cultures was enhanced following laser exposure.

Transplantation of corneal stem cells cultured on amniotic membrane for corneal burn: experimental and clinical study

OBJECTIVE: To investigate the proliferation and differentiation of cultured corneal stem cells and determine the effect of corneal stem cells cultured on amniotic membranes on the limbal area for treating corneal burns. METHODS: The proliferation and differentiation of corneal stem cells in vitro had been examined using colony-forming efficiency and immunohistochemistry. The stem cells had been cultured on amniotic membranes and transplanted to the limbal area for treating corneal burns. RESULTS: Corneal stem cells had a high proliferation capacity in primary and first passage, cytokeratin 3 was not expressed in primary culture but partly in first passage. The stem cells could proliferate to form cell layer on an amniotic membrane. When transplanted, stem cells could survive on limbus. After transplantation, ocular inflammation resolved, the cornea re-epithelialized, the stromal opacity reduced, the superficial neovascularity was lessened and the conjunctival fornix re-established. CONCLUSIONS: Ocular surface conditions could be improved by allograft of corneal stem cells cultured on amniotic membranes.

Expression of adenosine receptors in human retinal pigment epithelium cells in vitro

Background Adenosine receptors （ADORs） have been reported to play a role in experimental myopia. This study aimed to determine the distribution of ADORs in human retinal pigment epithelium （RPE） cells cultured in vitro.Methods Human RPE cells （cell line D407） were cultured in vitro. ADOR mRNA in RPE was detected by reverse transcription polymerase chain reaction. ADOR protein expression in RPE was confirmed by Western blotting analysis of cell lysates. Confocal fluorescence microscopy was used to study the subcellular distribution of ADORs.Results All four subtypes of ADORs mRNA and protein were expressed in human RPE. This was confirmed by Western blotting analysis. The ADOR subtypes were differently distributed within the cells. ADORA1 was expressed in nucleus, perinucleus and cytoplasm of RPE. ADORA2A was concentrated mainly in one side of the perinucleus and cytoplasm of RPE. ADORA2B was strongly expressed in the nucleus, perinucleus and the cytoplasm, and ADORA3 was expressed weakly in the cytoplasm of RPE.Conclusions ADORs are expressed in human RPE. The different distribution at the subcellular level suggests different functions of ADOR subtypes.

体外原代培养人视网膜色素上皮细胞(英文)

目的:探讨建立体外原代培养人类视网膜色素上皮细胞(RPE)技术。为研究溶血磷脂酸(lysophos-phatidic acid, LPA)对培养的人类视网膜色素上皮细胞DNA合成与增殖的作用。方法:取自愿者贡献的意外事故成年眼球,用2.5g/L胰酶消化获取人RPE细胞、150mL/L胎牛血清的DMEM培养液培养,细胞接近融合状态时进行传代培养。取自愿者贡献的眼球并游离其视网膜色素上皮细胞,用DMEM加100mL/L血清及MEM氨基酸和庆大霉素,进行细胞传代培养。用溶血磷脂酸、转移因子β2(TGF-β2)及LPA+TGF-β2进行视网膜色素上皮细胞增殖试验,用细胞染色法进行细胞计数,提取视网膜色素上皮细胞DNA,用Spectrophotometer进行DNA定量测定。结果:RPE原代细胞镜下为圆形,大小不一,内含较多的色素颗粒,胞核无法辨认。原代细胞培养贴壁后3d增殖速度明显加快,至4～5d即可基本融合,细胞浆内色素颗粒则随传代次数增多而逐渐减少。第3代培养的视网膜色素上皮细胞膜表面可见微绒毛,细胞质内细胞器丰富,线粒体量多,体积较小,内外膜分界清晰,嵴较短。色素颗粒散在分布于胞浆内,多数细胞质内数量较少呈高电子密度包含物。LPA对原代培养的视网膜色素上皮细胞增殖有促进作用。含有和/或缺乏10mL/L小牛血清的两种LPA(10μmol/L雪均明显刺激视网膜色素上皮细胞?

细胞因子及苏拉明对视网膜色素上皮细胞增殖的影响

目的:研究在血小板源性生长因子(PDGF)和白介素-1β(IL-1β)作用下苏拉明(suramin)对培养的人视网膜色素上皮(retinapligmentepithel-um,RPE)细胞增殖的影响。方法:将不同浓度的(15,150,250mg/L)苏拉明分别加入用PDGF10μg/L或IL-110μg/L培养的RPE细胞培养液中,继续培养3d后采用四甲基偶氮唑盐(tetrazoliu,mMTT)比色法,细胞分裂指数计数和核仁组成区嗜银染色(AgNORs)检测在细胞因子作用下不同浓度苏拉明对RPE增殖活力的影响。结果:含有PDGF10μg/L或IL-1β10μg/L的培养液显著刺激RPE的增殖,苏拉明明显抑制了这两种细胞因子条件下RPE的增殖,在150mg/L时的抑制率分别为26%和18%,对细胞的形态无明显影响。结论:苏拉明对PDGF或IL-1β刺激的RPE的增殖有显著抑制作用,该作用为非毒性作用。进一步证明苏拉明对增生性玻璃体视网膜病变(prolifera-tivevitroeretinopath,PyVR)中相关细胞因子的拮抗作用,为临床防治PVR提供了新的用药思路。

人参二醇组皂甙对人视网膜色素上皮细胞增生的抑制作用

目的 :研究人参二醇组皂甙 ( panaxadiol saponins,PDS)对体外培养视网膜色素上皮( RPE)细胞增生的直接抑制作用及可能机制。方法 :通过活细胞计数法与 MTT比色法分别检测不同浓度的 PDS和 2 0 0 mg· L-1PDS在不同作用时间 ( 6～ 1 2 0 h)对 RPE细胞增生及代谢的影响。结果 :PDS组细胞增殖明显受抑并出现细胞脱落 ,40 0 mg· L-1PDS具有最强的抑制效应 ,其明显抑制效应在 6h出现 ,96h达高峰。PDS组 A值明显低于对照组 ,RPE细胞生存率下降。结论 :PDS可能通过阻滞钙通道降低细胞内钙浓度、干扰 RPE细胞代谢 ,对

姜黄素对IL-1β诱导的兔RPE细胞中核因子-κB相关炎性因子表达的抑制作用

背景白细胞介素-1β（IL-1β）是增生性玻璃体视网膜病变（PVR）早期释放的重要炎性因子，研究证实姜黄素能抑制IL-1β诱导的兔视网膜色素上皮（RPE）细胞的增生，但其是否能够对PVR发挥抗炎作用尚不清楚。目的观察姜黄素对IL-1β诱导的兔RPE细胞移行的影响，探讨姜黄素对IL-1β诱导的RPE细胞中炎性因子表达的影响。方法采用对数生长期原代培养的第4代兔RPE细胞进行实验，在无血清DMEM培养基中分别添加0、0．1、1．0和10．0μg／L的IL-1β作用于细胞24h，分别采用Western blot和逆转录PCR法检测细胞中环氧合酶-2（COX-2）蛋白和mRNA的表达以筛选IL-1β最佳质量浓度。将培养的RPE细胞分为IL-1β组和姜黄素＋IL-1β组，分别在无血清培养基中添加1．0μg／L IL-1β和1．0μg／L IL-1β联合10μg／ml姜黄素作用于细胞24、48和72h，仅用无血清培养基培养的细胞作为对照组。培养的细胞行苏木精-伊红染色，于光学显微镜下计算进入损伤区的细胞数，比较各组细胞的移行能力；分别采用Western blot法和逆转录PCR法检测各组RPE细胞中COX-2蛋白及其mRNA的相对表达量，采用Westernblot法检测并比较各组细胞中核因子-κB p65（NF—κB p65）蛋白和核因子κB抑制蛋白-α（IκB—α）的相对表达量；采用免疫化学染色法检测NF—κB p65、IκB-α和COX-2在各组RPE细胞中的表达和定位。结果初分离的兔RPE细胞呈球形，细胞中可见大量黑色素颗粒；第4代细胞色素颗粒明显减少，接近融合的细胞形态为长梭形，呈拉网状分布。免疫细胞化学染色法结果显示，细胞角蛋白（AE1／AE3）在细胞质呈阳性表达。对照组在培养24、48和72h移行的细胞数分别为（31．93±1．21）、（36．27±2．50）和（38．33±2．40）个，IL-1β组分别为（45．73±2．30）、（71．13±1．92）和（80．60±1．71）个，而姜黄素＋IL-1β组分别为（13．13±2．20）、（14．93±1．10）和（12．60±1．51）个，各时间点IL-1β组细胞移行细胞数均明显高于对照组，而姜黄素＋IL-1β组移行细胞数均明显低于对照组和IL-1β组，差异均有统计学意义（均P〈0.05）。IL-1β质量浓度为1．0μg/L时，RPE细胞中COX-2蛋白及其mRNA的相对表达量达峰，以1．0μg／L IL-1β的剂量进行后续实验。细胞培养后24、48和72h，姜黄素＋IL-1β组细胞中COX-2蛋白及其mRNA的相对表达量均明显低于IL-1β组，差异均有统计学意义（均P〈0.05）。IL-1β作用于细胞后48h，细胞中NF—κB p65蛋白的相对表达量最高，与IL-1β组相比，各时间点姜黄素＋IL-1β组细胞中NF—κB p65蛋白相对表达量降低，差异均有统计学意义（均P〈0．05）。IL-1β组药物作用于细胞后48h细胞中IκB—α降至最低，各时间点姜黄素＋IL-1β组细胞中IκB-α值均明显高于IL-1β组，差异均有统计学意义（均P〈0.05）。免疫细胞染色结果显示，IL-1β组RPE细胞的细胞核及细胞质中NF—κB p65呈强阳性表达，对照组表达较弱，姜黄素＋IL-1β组细胞中NF—κB p65的表达强度较IL-1β组明显降低；与对照组相比，IL-1β组细胞的细胞质中IκB-α表达强度明显减弱，而COX-2表达明显增强；与IL-1β组比较，姜黄素＋IL-1β组细胞中IκB-α表达明显增强，而COX-2表达明显减弱。结论姜黄素可抑制IL-1β引起的兔RPE细胞的移行，IL-1β通过激活NF—κB信号通路刺激细胞中COX-2的表达，姜黄素可通过阻断这一途径而抑制炎性因子的表达，从而发挥抗炎作用。

苏拉明对体外培养视网膜色素上皮细胞增殖的影响

目的 研究苏拉明 (suramin)对培养人视网膜色素上皮细胞 (retinal pigm ent epithelium,RPE)增殖的影响 .方法 将不同质量浓度的苏拉明 (1.5 ,15和 15 0 mg· L- 1 )加入RPE细胞培养液 ,采用四甲基偶氮唑盐 (tetrazolium,MTT)比色法 ,细胞分裂指数计数和核仁组成区嗜银染色(Ag NORs)检测苏拉明对 RPE增殖活力的影响 .结果 含有10 0 m L· L- 1小牛血清的培养液可以显著刺激 RPE的增殖(P<0 .0 1) ,无血清组 A值为 0 .19± 0 .0 1、含血清组 A值为0 .30± 0 .0 1;苏拉明抑制了 10 0 m L· L- 1血清条件下 RPE的增殖 ,呈剂量依赖性 ,最大抑制率达 5 1% ,3种浓度组 A值分别为 0 .2 9± 0 .0 1,0 .2 4± 0 .0 1和 0 .14± 0 .0 1,对细胞的形态无明显影响 .结论 苏拉明对血清刺激

人脂肪间充质干细胞向视网膜色素上皮样细胞的诱导分化及其在体应用研究

背景 目前,干细胞疗法治疗视网膜变性疾病是眼科研究的热点之一.研究已证实骨髓间充质干细胞（MSCs）可以体外诱导分化为视网膜色素上皮（RPE）样细胞,但由于取材困难,临床应用受到了一定的限制.人脂肪间充质干细胞（ADSCs）与MSCs有类似的特性且容易获取,但人ADSCs能够诱导分化为RPE细胞的研究尚不多见. 目的 评估人ADSCs向RPE样细胞诱导分化的可行性及其在体应用的安全性.方法 将体外培养的第3代人ADSCs接种于6孔板进行培养,12h后于实验组培养液中加入100 ng/ml表皮生长因子（EGF）、50 μmol/L牛磺酸和5×10-7 mol/L视黄酸进行诱导,常规培养的细胞作为对照组.采用RPE细胞标志物Pan细胞角蛋白（Pan-CK）单克隆抗体进行免疫荧光检测,对诱导的细胞进行鉴定,采用细胞膜示踪剂PKH26标记法示踪诱导细胞,将诱导后的RPE样细胞悬液lμl注入6只BALB/c裸鼠的右眼玻璃体腔,另6只裸鼠玻璃体内注射等容量PBS.于注射后1个月摘取实验动物右眼眼球,各组中3只眼球用于组织病理学检查,在光学显微镜下观察玻璃体视网膜的形态学改变,另3只眼球在透射电子显微镜下观察玻璃体视网膜超微结构的改变,评估诱导细胞在体应用的安全性. 结果 体外培养的第3代人ADSCs呈细长多角形,生长良好.对照组细胞Pan-CK表达缺失,而诱导细胞的细胞膜Pan-CK表达阳性,呈红色荧光.诱导的细胞经PKH26标记后细胞膜呈红色荧光.诱导的细胞悬液于裸鼠玻璃体内注射后1个月,光学显微镜下可见诱导细胞位于视网膜表面,视网膜各层组织结构排列清晰;透射电子显微镜下可见视网膜神经节细胞（RGCs）细胞膜完整,线粒体结构正常,染色质均匀. 结论 人ADSCs体外诱导后可分化为RPE样细胞,PKH26可对诱导后细胞进行细胞示踪,分化的细胞玻璃体腔注射后短期内未发现视网膜的不良反应。

兔虹膜和视网膜色素上皮细胞的体外培养比较

目的 观察比较有色素兔虹膜色素上皮(IPE)及视网膜色素上皮(RPE)细胞的体外生长状况。方法采用酶消化法及酶辅助机械分离法分别分离IPE和RPE细胞。观察培养的两种细胞的生长状况,并对其进行免疫组化鉴定。结果原代IPE及RPE细胞大多呈圆形或六边形,细胞内充满了黑色素颗粒。随着传代的增加,呈梭形或纤维细胞样生长的细胞增多,细胞中的色素颗粒也逐渐减少。细胞角蛋白免疫组化染色显示IPE及RPE细胞绝大多数呈棕黄色阳性反应。Desmin免疫组化染色显示IPE细胞中阳性细胞约占5％。 结论 分离培养的IPE和RPE细胞纯度很高,IPE细胞中绝大多数为后层IPE细胞。IPE和RPE细胞的形态及体外生长特点类似。

人视网膜色素上皮细胞的培养

目的 :培养人视网膜色素上皮细胞 ,为采用视网膜色素上皮移植治疗色素变性奠定基础。方法 :用胰酶消化法获取人胎儿视网膜色素上皮细胞进行原代及传代培养。角蛋白抗体免疫细胞化学染色鉴定细胞。结果 :视网膜色素上皮细胞在体外生长良好 ,胞浆内含有丰富的色素颗粒。抗角蛋白反应阳性。结论

兔眼与猪眼视网膜色素上皮细胞分离及培养方法的对比

目的分别对兔眼及猪眼视网膜色素上皮（RPE）细胞进行体外分离、培养和鉴定，探讨二者的异同点及注意事项。方法对有色兔眼和猪眼的RPE细胞采用胰蛋白酶消化法进行分离、培养和传代，取第4代细胞用于实验。免疫细胞化学染色对传代的细胞进行鉴定。光学显微镜下观察并比较培养的2种动物来源RPE细胞的形态、特征，并对2种动物来源的RPE细胞的培养过程进行比较。结果2种动物来源的RPE细胞分离方法的不同与各自眼球的解剖结构相关，主要体现在消化过程，猪眼RPE细胞1次消化即可分离，兔眼RPE细胞需2次消化得到。原代猪眼RPE细胞24h内贴壁较兔眼48～72h贴壁时间早。培养早期细胞生长活跃，随传代次数增加活力下降，色素颗粒减少。免疫细胞化学染色提示细胞角蛋白（AE1／AE3）表达阳性。结论2种动物来源的RPE细胞均可通过胰蛋白酶消化法分离，获得的RPE细胞数量多，培养成功率高。猪眼RPE细胞的分离培养更为容易。4代以内的RPE细胞适合于实验研究。

人参皂甙Rb_2对培养人视网膜色素上皮细胞增生的抑制作用

目的 研究人参皂甙Rb2 (Ginsenoside Rb2 )对体外培养视网膜色素上皮细胞增生的直接抑制作用及可能机制。方法 通过活细胞计数法与MTT比色法分别检测不同浓度 (2 5 0、2 0 0、15 0、12 0、10 0、5 0、10 μg ml)Rb2 和Rb215 0 μg ml在不同作用时间 (6～ 12 0h)对RPE细胞增生及代谢的影响。 结果 Rb2 组细胞增殖受到抑制并出现细胞脱落 ,Rb2 2 0 0 μg ml具有最强的抑制效应 ,其明显抑制效应在 6h即出现 ,96h达高峰。Rb2 组A值明显低于对照组 ,RPE细胞生存率下降。结论 Rb2 可能通过阻滞钙通道降低细胞内钙浓度。

Transwell小室共培养条件下缺氧时视网膜色素上皮细胞对内皮细胞增殖的影响

目的研究在Transwell小室共培养系统中,缺氧时视网膜色素上皮细胞(retinal pigment epithelium,RPE)对血管内皮细胞增殖的影响。方法培养并鉴定RPE细胞和人脐静脉血管内皮细胞,利用200μmol/LCoCl2造成细胞缺氧、Transwell小室建立细胞共培养模型。实验分为4组:对照组:内皮细胞;共培养组:内皮细胞+RPE细胞;缺氧对照组:内皮细胞+CoCl2;缺氧共培养组:内皮细胞+RPE细胞+CoCl2,各组分别培养0 h2、4 h4、8 h后,应用MTT法检测内皮细胞增殖情况。结果常氧时,对照组内皮细胞光吸收值(A值)在24 h达最高;对照组和共培养组内皮细胞光吸收值在各时间点差别均无统计学意义(P>0.05)。缺氧条件下,缺氧对照组A值在24 h4、8 h均低于对照组(P<0.05);缺氧对照组A值在24 h、48 h低于缺氧共培养组(P<0.05)。结论缺氧早期内皮细胞增殖受到抑制;缺氧条件下,RPE促进内皮细胞的增殖。