内质网应激在氧化低密度脂蛋白诱导的人RPE细胞凋亡中的作用

目的：观察内质网应激(ERS)在氧化低密度脂蛋白(OxLDL)诱导的人视网膜色素上皮(RPE)细胞凋亡中的作用。

方法：人RPE细胞系ARPE19采用低糖DMEM培养基和10%胎牛血清进行常规培养。实验分为3组：对照组(常规培养的ARPE19)、OxLDL组(加入5、10、25、50、100μg/mL OxLDL)和LDL组(加入5、10、25、50、100μg/mL LDL)培养24h。分别采用细胞计数试剂盒(CCK8)检测各组细胞活性,流式细胞仪检测凋亡比例,蛋白质印迹(Western blotting)检测ERS相关蛋白及凋亡相关酶的表达。激光共聚焦显微镜观察人RPE细胞吞噬红色荧光探针Dil标记的氧化低密度脂蛋白(Dil-OxLDL)情况。

结果：CCK8结果显示：对照组细胞存活率为(100±5.637)%,加入5、10、25、50、100μg/mL OxLDL后细胞活力分别为(105.298±9.395)%、(97.106±5.417)%、(77.015±4.055)%、(67.613±3.853)%和(43.872±9.532)%(P<0.05); 加入5、10、25、50、100μg/mL LDL后细胞活力分别为(97.55±6.217)%、(99.640±3.586)%、(90.495±2.786)%、(83.552±9.171)%和(90.910±1.429)%(P>0.05)。流式细胞仪结果显示浓度为25μg/mL的OxLDL会明显诱导细胞凋亡,对照组、OxLDL组(25μg/mL)和LDL(25μg/mL)组凋亡率分别是(5.271±0.519)%、(41.23±1.686)%和(13.07±2.579)%(P<0.01); Western blotting结果显示OxLDL(25μg/mL)组的ERS相关蛋白和凋亡相关酶的表达量明显高于对照组与LDL组(Caspase-12：F=50.53, P<0.05; GRP78：F=55.60, P<0.05; CHOP：F=38.22, P<0.05; XBP-1：F=53.94, P<0.05; ATF6：F=20.01, P<0.05),而LDL组(25μg/mL)和对照组之间无差异(P>0.05)。

结论：ERS参与了OxLDL诱导的人RPE细胞凋亡,调控ERS可能抑制人RPE细胞凋亡,从而治疗RPE细胞凋亡相关疾病。     

盐酸甲氯芬酯对人视网膜色素上皮细胞增生的影响

目的观察盐酸甲氯芬酯对体外培养的人视网膜色素上皮细胞增生的影响。方法通过MTT比色法和核仁嗜银蛋白染色分析,观察盐酸甲氯芬酯对人视网膜色素上皮细胞增生的影响。结果不同浓度盐酸甲氯芬酯可以促进人视网膜色素上皮细胞增生,在10~1000mg·L-1的范围内呈剂量效应关系,并随时间的延长,促进效应增强。嗜银蛋白染色结果:10mg·L-1盐酸甲氯芬酯作用24h后其胞核内AgNORs的数量增加,平均数为2.0(P=0.029);100mg·L-1盐酸甲氯芬酯作用12h即有胞核内AgNORs的增多,平均数为2.0(P=0.029)。随剂量的增加和时间的延长,胞核内AgNORs的数量增加。结论盐酸甲氯芬酯可以促进人视网膜色素上皮细胞增生,并与剂量和作用时间有一定的关系。     

Enhanced expression of glutathione-S-transferase A1-1 protects against oxidative stress in human retinal pigment epithelial cells

Glutathione-S-transferases (GSTs) play an important role in protection mechanisms against oxidative stress. We sought to determine whether over-expression of human GSTA1-1 in RPE cells is able to attenuate H(2)O(2)-induced oxidative stress. SV40-transformed human fetal RPE cells were stably transfected with pRC/hGSTA1-1 vector which carries a full-length of human GSTA1-1 cDNA. The control RPE cells were either non-transfected or transfected with control vector pRC. Expression of hGSTA1-1 protein in these cells was confirmed by Western blot and immunocytochemical analyses. The protective effects of hGSTA1-1 on cell viability and mitochondrial DNA (mtDNA) damage caused by H(2)O(2) were examined with MTT assay and quantitative PCR (QPCR), respectively. The hGSTA1-1 transfected RPE cells exhibited a similar morphology and growth rate as control RPE cells. Immunocytochemical analysis showed robust expression hGSTA1-1 in hGSTA1-1 transfected cells versus background staining in control cells. Western blotting of protein extracts from cells transfected with hGSTA1-1 revealed a 26 kDa protein band which corresponds to the size of recombinant mature hGSTA1-1. The active GST present in the hGSTA1-1 transfected cells was approximately three times higher than in control cells. The MTT assay showed a significantly greater viability of hGSTA1-1 cells in response to H(2)O(2) (100 and 200 microm) compared to control cells (p<0.05). QPCR indicated that mtDNA damage was significantly decreased in hGSTA1-1 cells than in control cells (p<0.05). Human GSTA1-1 transfection protect against RPE cell death and mtDNA damage caused by H(2)O(2), suggesting an important role of GST in protection against oxidative stress in RPE cells.     

视网膜色素上皮细胞氧化应激相关microRNA的鉴定

目的：应用miRNA表达谱芯片筛选视网膜色素上皮细胞与氧化应激相关的miRNA,为更加全面深入地研究年龄相关性黄斑病变(age-related macular degeneration,AMD)发生的分子机制提供新的思路。

方法：培养D407细胞,分别使用100、200、400μmol/L H₂O₂处理细胞24h后,Trizol试剂抽提细胞总RNA,使用Exiqon miRCURY LNA^TM microRNA表达谱芯片(miRBase 16.0数据库)检测不同浓度处理后D407细胞miRNA的表达差异,并将不同浓度处理后的变化进行聚类分析。使用Stem loop realtime PCR对芯片结果进行验证,并运用生物信息学方法预测差异miRNA调控的靶基因。

结果：芯片所包含的1 425个已知miRNA中,共有367个在不同浓度H₂O₂处理后表达发生变化。通过Treeview软件进行聚类分析发现miR-31等17个miRNA随H₂O₂浓度的升高呈现逐渐降低的趋势,miR-206等7个则逐渐升高。PCR验证显示芯片结果准确性较好。

结论：H₂O₂作用前后RPE的miRNA表达存在明显差异,miRNA作为转录后水平的调节分子,参与了细胞氧化应激反应,可能在AMD的发生发展中起有重要作用。     

黄酮对视网膜色素上皮细胞氧化损伤的保护作用

目的:观察黄酮对氧化剂所诱导的视网膜色素上皮细胞(RPE)的保护作用。方法:在体研究中,预先给予5g/L黄酮滴眼液(3次/d),1wk后舌下静脉注射NaIO3诱导大鼠RPE变性,在2和4wk末,采用视网膜电图(ERG)测量C波。离体研究中,采用缺氧、H2O2、NaN3和t-BHP诱导RPE细胞损伤,并用MTT法检测细胞的存活率。结果:ERG的C波结果表明,第4wk末,黄酮抑制了由NaIO3诱导的大鼠RPE变性。离体研究结果表明,黄酮对多种氧化剂所诱导的RPE细胞损伤具有保护作用。结论:黄酮对氧化诱导的在体和离体视网膜色素上皮细胞均具有保护作用。     

Effects of flavone on the oxidation-induced injury of retinal pigment epithelium cells

AIM: To investigate the effect of flavone on oxidation- induced injury in retinal pigment epithelium cells. METHODS: In in vivo studies, NaIO3-induced RPE degene- ration in rat eyes was treated with 0.5% flavone eye drops 3 times a day for 1 week before and 4 weeks after NaIO3 injection. At the end of 2 and 4 weeks, all rats were measured c-wave by electroretinogram (ERG). In in vitro studies, ARPE-19 cells were treated with hypoxia, H2O2, NaN3 and t-BHP to induce cell damages. MTT assay was used to measure the viable cells. RESULTS: The ERG c-wave results showed that flavone reversed NaIO3-induced injury at the end of 4 weeks. In vitro results showed flavone reversed the various oxidants-induced injuries in RPE cells. CONCLUSION: Flavone could prevent the RPE from oxidation- induced injury both in vivo and in vitro.     

干性ARMD中视网膜色素上皮细胞的作用及损伤机制

年龄相关性黄斑变性(age-related macular degeneration,ARMD)是导致老年人中心视力不可逆丧失的主要原因。ARMD的典型特征是视网膜色素上皮(retinal pigment epithelium,RPE)和脉络膜毛细血管发生退行性改变及黄斑区出现玻璃膜疣。临床上ARMD分为两种亚型：非渗出型(干性或萎缩型)和渗出型(湿性或新生血管型)。该病的发生是年龄、环境、遗传、吸烟、氧化应激和心血管功能障碍等多种因素相互作用的结果。鉴于RPE细胞在ARMD发病中的重要作用,现以RPE细胞为重点,总结了蓝光、吸烟、氧化应激、脂褐素积累、慢性炎症和蛋白质稳态对干性ARMD发病的作用和可能机制,为认识和预防干性ARMD的发生提供新的帮助。     

贝伐单抗对视网膜色素上皮细胞抗氧化功能的影响

目的 观察贝伐单抗对人视网膜色素上皮(retinal pigment epithelium,RPE)细胞抗氧化功能的影响,以探讨抗新生血管内皮生长因子(vascular endothelial growth factor,VEGF)制剂治疗年龄相关性黄斑变性后黄斑部萎缩的可能机制.方法 用含终浓度0.25g·L-1贝伐单抗的DMED/F12培养液培养人RPE细胞系ARPE-19细胞,根据处理时间的不同分为Oh组(对照组)、12 h组、24 h组、48 h组、72 h组共5组,加入H2O2诱导氧化应激反应.用CCK-8法检测细胞活性,MitoSox Red荧光染色检测细胞内线粒体活性氧(reactive oxygen species,ROS)产生水平,JC-1荧光染色检测细胞线粒体膜电位的变化;分别用逆转录聚合酶链反应(RT-PCR)及免疫蛋白印迹法(Western blot)检测各组促氧化因子NADPH氧化酶4(NADPH oxidase 4,NOX4)和抗氧化因子血红素氧合酶-1(heme oxygenase 1,HO-1) mRNA和蛋白的表达水平.结果 CCK-8检测结果显示:上述处理对细胞活性无显著影响,Oh组、12 h组、24h组、48h组和72 h组细胞活性分别为(100.2±3.3)％、(99.2±2.7)％、(102.5±6.4)％、(103.9±3.7)％和(103.6±3.3)％,差异无统计学意义(P＞0.05);与对照组相比,12 h、24h、48 h、72 h组细胞内ROS水平上升,差异有统计学意义(P＜0.05);线粒体膜电位在12 h、24h、48 h、72 h组均较对照组降低,差异有统计学意义(P＜0.01),48 h达最低,72 h时显著提高,但仍低于对照组.RT-PCR和Western blot检测结果显示:与对照组比较,NOX4 mRNA和蛋白的表达在12 h、24h、48 h和72 h组均上升,而且在24h表达最高,之后明显下降,但仍高于对照组,差异有统计学意义(P＜0.01).与对照组比较,HO-1 mRNA的表达在24h、48 h和72 h组均下降,而HO-1蛋白的表达在48 h和72 h组下降,差异有统计学意义(P＜0.05).结论 临床浓度的贝伐单抗可以降低RPE的抗氧化功能,可能是长期抗VEGF治疗后黄斑部进行性萎缩的原因之一.     

干性年龄相关性黄斑变性患者视网膜色素上皮细胞损伤机制

年龄相关性黄斑变性(age-related macular degeneration,AMD)是中老年人视力障碍的重要原因之一。AMD分为2型:萎缩型(干性)和渗出型(湿性)。本文从光照损伤、脂褐素积累、氧化损伤、内质网应激4个方面,综述了干性AMD研究中基于视网膜色素上皮细胞损伤的相关研究进展,为进一步探索其发病机制提供依据和方向。     

紫外线B照射在抑制人视网膜色素上皮细胞自噬中的作用

目的　探讨紫外线B(ultraviolet B,UVB)照射在抑制人视网膜色素上皮细胞株ARPE-19细胞自噬中的作用。方法　不同剂量UVB照射ARPE-19细胞后不同时间收集细胞,应用MTT法检测UVB照射对细胞存活的影响;Western blot检测细胞中自噬蛋白LC3的表达及LC3-II/LC3-I比率、Beclin-1蛋白表达水平;应用细胞自噬增强剂雷帕霉素（rapamycin,RAPA）和细胞自噬抑制剂氯化铵（NH₄Cl）进一步验证UVB照射对于细胞自噬的调节;采用环氧霉素（epoxomicin,EPO）检测泛素-蛋白酶体系统(ubiquitin-proteasome system,UPS)对于UVB作用的影响。MTT法检测细胞自噬变化对于细胞生存的影响。结果　UVB照射组诱导细胞剂量依赖性的死亡,100 mJ·cm^-2 UVB照射后24 h细胞出现明显死亡,和对照组相比差异具有统计学意义（P<0.05）;200 mJ·cm^-2 UVB照射后24 h和对照组相比差异有显著统计学意义(P<0.01);两种剂量的UVB照射后48 h细胞有一定程度的恢复。和对照组相比,UVB照射组ARPE-19细胞中LC3-II/LC3-I比率和Beclin-1蛋白表达水平均降低（均为P<0.05)。和UVB照射组相比,UVB+RAPA组ARPE-19细胞中明显升高了LC3-II/LC3-I比率,UVB+NH₄Cl组进一步降低了LC3-II/LC3-I比率,RAPA和NH₄Cl在一定程度上恢复了UVB照射降低的Beclin-1蛋白表达水平（均为P<0.05)。和对照组相比,EPO组明显增加了ARPE-19细胞中LC3-II/LC3-I比率、升高了Beclin-1蛋白表达水平（均为P<0.05)。与UVB照射组相比,UVB+EPO组ARPE-19细胞中LC3-II/LC3-I比率无明显改变(P>0.05),而Beclin-1蛋白表达水平差异有统计学意义(P<0.05)。与对照组相比,RAPA组细胞死亡率增多,两组间差异有统计学意义（P<0.05）,NH₄Cl组细胞无明显死亡,与UVB照射组相比,UVB+RAPA组和UVB+NH₄Cl组细胞死亡率均无明显改变（均为P>0.05）。细胞自噬的改变不能逆转UVB诱导的ARPE-19细胞死亡。结论　UVB照射抑制了ARPE-19细胞自噬的启动及自噬流,UPS的改变不影响UVB对于自噬流的抑制。UVB照射可通过减少RPE细胞、损害细胞自噬能力增加RPE细胞变性,增加年龄相关性黄斑变性发生的风险。     

Lycium barbarum polysaccharides protected human retinal pigment epithelial cells against oxidative stress-induced apoptosis

AIM: To investigate the protective effect and its mechanism of lycium barbarum polysaccharides (LBP) against oxidative stress-induced apoptosis in human retinal pigment epithelial cells. METHODS: ARPE-19 cells, a human retinal pigment epithelial cell lines, were exposed to different concentrations of H2O2 for 24h, then cell viability was measured by Cell Counting Kit-8 (CCK-8) assay to get the properly concentration of H2O2 which can induce half apoptosis of APRE-19. With different concentrations of LBP pretreatment, the ARPE-19 cells were then exposed to appropriate concentration of H2O2, cell apoptosis was detected by flow cytometric analysis. Expression levels of Bcl-2 and Bax were measured by real time quantitative polymerase chain reaction (RT-PCR) technique. RSULTS: LBP significantly reduced the H2O2-induced ARPE-19 cells’ apoptosis. LBP inhibited the H2O2-induced down-regulation of Bcl-2 and up-regulation of Bax. CONCLUSION: LBP could protect ARPE-19 cells from H2O2-induced apoptosis. The Bcl-2 family had relationship with the protective effects of LBP.     

巴曲酶对视网膜色素上皮细胞增生活性的影响

目的探讨巴曲酶对视网膜色素上皮细胞(retinal pigment epithelium,RPE)增生活性的影响。方法利用体外培养的RPE细胞系,应用噻唑兰比色法观察不同浓度的巴曲酶对RPE增生活性的影响。结果当巴曲酶浓度不大于1/10KU.mL-1时各浓度巴曲酶组(1/100KU.mL-1、1/32KU.mL-1、1/16KU.mL-1、1/10KU.mL-1)与对照组吸光度(optical density,OD)值差异无统计学意义(P=0.068、0.455、0.675、0.159);巴曲酶浓度为1/8KU.mL-1时OD值明显降低(P=0.000)。OD值与培养液渗透压呈明确的负相关(r=-0.951,P=0.000);巴曲酶组和等渗对照组OD值的差异没有统计学意义(P=0.749)。结论渗透压符合RPE生长要求的前提下,巴曲酶对体外培养的RPE增生无明显影响,其应用于玻璃体手术中控制眼内出血的可行性值得进一步研究。     

Bcl-2 overexpression increases survival in human retinal pigment epithelial cells exposed to H(2)O(2)

The integrity of the retinal pigment epithelium, especially that of the macula is essential for the preservation of vision into old age. The chronic exposure to sunlight and peroxidized lipids from phagocytized photoreceptor outer segments imposes a high level of oxidative stress on the retinal tissues, which increases with age as antioxidant protection declines and therefore could accelerate apoptosis. Bcl-2 known to facilitate mitochondrial DNA repair and cellular survival in other tissues was overexpressed in a single clone of human retinal pigment epithelium cells after stable transfection with humanbcl-2 in rhoSFV-neoexpression factor. Near confluent cells (2nd-4th generation permanently bcl-2 transfected) were protected from mitochondrial dysfunction after exposure to H(2)O(2) up to 150 microM. With 200 microM H(2)O(2), function in transfected cells declined by only 25% control activity as determined by MTT reduction assays, compared to wild type and vector only transfected cells expressing normal bcl-2 levels. Similarly the bcl-2 -transfected cells were more resistant to mitochondrial DNA damage after H(2)O(2) treatment than the other groups and suffered 50% less damage after exposure to 200 microM H(2)O(2), as assayed by quantitative polymerase chain reaction assays. These data suggest that bcl-2 overexpression protects human RPE cells from mitochondrial respiratory dysfuction, mitochondrial DNA damage and promotes cellular survival in response to oxidative stress induced by H(2)O(2).     

Effects of lipid peroxidation products on lipofuscinogenesis and autophagy in human retinal pigment epithelial cells

Several lines of evidence suggest that progressive dysfunction of the retinal pigment epithelium (RPE) is central to the pathogenesis of age-related macular degeneration (AMD). We previously demonstrated that protein modifications with lipid peroxidation products, such as 4-hydroxynonenal (HNE) and malondialdehyde (MDA), induce lysosomal dysfunction in RPE cells in vitro. Here, we investigated whether phagocytosis of modified photoreceptor outer segments (POS) affects lipofuscinogenesis and autophagy, two interrelated processes directly connected to lysosomal function. Incubation of human RPE cells with HNE- and MDA-modified POS resulted in pronounced intracellular accumulation of granular material with lipofuscin-like autofluorescence. After daily treatment with modified POS for 7 days, cellular autofluorescence increased 8.2-fold as quantified by flow cytometry. In the presence of the lysosomal inhibitor ammonium chloride, unmodified POS likewise induced an 8.0-fold increase in autofluorescence. Spectral profiles of cellular autofluorescence after incubation with modified POS were unchanged compared to incubation with native POS. Autophagy activity, measured as turnover of metabolically radiolabeled endogenous proteins, was reduced by both HNE- and MDA-modified POS by 40%. Autophagy inhibition by 3-methyladenine and lysosomal inhibition by ammonium chloride induced lipofuscinogenesis even in the absence of POS. In summary, our results demonstrate that induction of lysosomal dysfunction by lipid peroxidation-derived protein modifications results in increased lipofuscinogenesis and reduced autophagy activity in RPE cells in vitro. These mechanisms may contribute to RPE cell dysfunction and degeneration in AMD.     

UV irradiation causes multiple cellular changes in cultured human retinal pigment epithelium cells

Background The retinal pigment epithelium maybe causally involved in the development and progression of age-related macula degeneration; however, the mechanisms leading to the development of age-related macula degeneration remain largely unknown. The purpose of this study was to examine cellular changes in the retinal pigment epithelium induced by direct irradiation with UV light in culture.Methods Retinal pigment epithelium cells from post-mortem human retinas were used to obtain dissociated cultures with cells retaining the ability to differentiate in vitro. These cells were cultured over several days to weeks. The UV radiation (UV-A and UV-B) occurred under sterile conditions with a 100 HBO/mercury bulb attached to a dissecting microscope, delivering co-axial illumination. The time dependence of irradiation effects was analysed using morphometric, immunohistochemical, functional and apoptosis-detecting techniques.Results Vital and proliferating retinal pigment epithelium cell cultures could be prepared consistently. The cells showed tissue-specific morphologies in vitro for several days to weeks. Pigment epithelium-derived factor was detected in these cells using immunocytochemistry and Western blots. The UV irradiation but not white light resulted in measurable alterations of cell shape and size. The irradiated cells showed partial swelling and shrinkage reminiscent of progressing apoptotic degeneration. TUNEL staining revealed that apoptosis was induced by UV light, but not detectably by white light. The phagocytosis of fluorescent micro-particles diminished after irradiation. These effects were dependent on the duration of irradiation.Conclusions Cultures of retinal pigment epithelium are suitable and sensitive models to study cell damage and may contribute to unravelling the pathogenetic mechanisms of retinal degeneration.     

单次注射康柏西普治疗渗出型AMD患者RPE隆起面积与容积变化

目的　观察单次玻璃体内注射康柏西普治疗渗出型年龄相关性黄斑变性（ａｇｅ-ｒｅｌａｔｅｄｍａｃｕｌａｒｄｅｇｅｎｅｒａｔｉｏｎ,ＡＭＤ）患者的视网膜色素上皮（ｒｅｔｉｎａｌｐｉｇｍｅｎｔｅｐｉｔｈｅｌｉｕｍ,ＲＰＥ）隆起的面积及容积变化,以评价康柏西普治疗ＡＭＤ的短期疗效及安全性。方法　选取渗出型ＡＭＤ患者４０例（４５眼）,所有患者均行玻璃体内注射康柏西普０．０５ｍＬ（０．５ｍｇ）治疗。术前、术后１周、１个月及３个月复诊行Ｃｉｒｒｕｓ５０００ＯＣＴ检查,比较治疗前后最佳矫正视力（ｂｅｓｔｃｏｒｒｅｃｔｅｄｖｉｓｕａｌａｃｕｉｔｙ,ＢＶＣＡ）、黄斑中心凹厚度及ＲＰＥ隆起的面积、容积。结果　术后１周、１个月,ＢＣＶＡ（ｌｏｇＭＡＲ）由术前１．２５±０．７９分别提升至０．９２±０．６６（Ｐ＜０．００１）和０．９４±０．６１（Ｐ＜０．００１）,３ｍｍ圆内ＲＰＥ隆起面积及容积由术前的（２．９１±１．７３）ｍｍ２、（０．５０±０．７３）ｍｍ３分别降至（２．７５±１．８２）ｍｍ２（Ｐ＝０．０２４）、（０．４２±０．７１）ｍｍ３（Ｐ＝０．０２０）和（２．３３±１．８５）ｍｍ２（Ｐ＝０．００２）、（０．３２±０．０９）ｍｍ３（Ｐ＝０．０４６）。术后３个月时ＢＣＶＡ下降至１．３０±０．８２,与术前差异无统计学意义（Ｐ＞０．０５）;３ｍｍ圆内ＲＰＥ隆起面积及容积增加到（２．７３±１．８１）ｍｍ２、（０．５１±０．７９）ｍｍ３,与术前相比差异均无统计学意义（均为Ｐ＞０．０５）。术后１周、１个月、３个月５ｍｍ圆内ＲＰＥ隆起面积及容积与术前差异均无统计学意义（均为Ｐ＞０．０５）。黄斑中心凹厚度仅在术后１个月由术前（２４４．５６±２５．３７）μｍ降至（２３４．９１±２１．５０）μｍ（Ｐ＝０．０４４）。所有患者均未出现严重眼部并发症及全身不良反应。结论　康柏西普短期内治疗渗出型ＡＭＤ可显著提高视力,恢复视网膜结构,具有良好的安全性。     

Induction of retinal pigment epithelium properties in ciliary margin progenitor cells

Purpose:  Degenerative processes in the retinal pigment epithelium (RPE) are known to play a pivotal role in the development of age-related maculopathy. Substitute RPE analogue cells could be used to preserve visual function. In this paper we investigate methods for the isolation, cultivation and RPE differentiation of undifferentiated cells from the ciliary marginal zone (CMZ) of rat eyes.
Methods:  The CMZ was isolated from enucleated rat eyes, cell spheres formed in serum-free suspension culture, Bromodeoxyuridine (BrdU) incorporation indicated mitotic activity. Following baseline differentiation status assessment, directional differentiation was induced by cultivating cells in RPE-conditioned medium and vasoactive intestinal peptide (VIP). The differentiation status was analysed by immunocytochemistry. Fluorescein isothiocyanate (FITC)-labelled latex beads were used for functional evaluation.
Results:  CMZ-derived cells were expanded for 6–12 months. Formation of spherical cellular conglomerates, subsphere formation and expression of nestin indicated progenitor cells. Baseline levels of markers MAP-2 for neuronal and GFAP for glial properties and baseline levels of bestrophin, cytokeratins 8 and 18 and RPE 65 for RPE properties were induced by serum culture, respectively. Culture in conditioned medium with addition of VIP significantly increased RPE marker expression and reduced neuronal features, uptake of latex beads indicated phagocytosis.
Conclusions:  We succeeded in isolating and cultivating cells from rodent CMZ with progenitor cell characteristics. Subsequently, these cells tested positive for neuronal, glial and RPE markers. Appropriate conditions significantly increased RPE marker expression. Unidirectional induction of differentiation makes the CMZ eligible as a source of regenerative ocular tissue for RPE-reconditioning therapy.     

H2O2对体外培养视网膜色素上皮细胞中BMP-6及其相关受体的影响

目的观察氧化应激对视网膜色素上皮(retinal pigment epithelium,RPE)细胞的损伤作用以及对骨形态发生蛋白(bone morphogenetic protein,BMP)-6及其相关受体的影响。方法 RPE细胞随机分为对照组:正常培养的RPE细胞;H₂O₂组:加入200μmol·L^-1H₂O₂处理的RPE细胞;实验组:加入200μmol·L^-1 H₂O₂和0.1 ng·L^-1 BMP-6的RPE细胞。采用活性氧(reactive oxygen species,ROS)检测试剂盒运用流式细胞仪检测对照组与H₂O₂组RPE细胞内ROS的变化,运用PCR及Western blot法检查对照组与H₂O₂组BMP-6基因及蛋白水平的变化;TUNEL染色法观察三组细胞凋亡的变化,并运用PCR及...     

Differential gene expression of early and late passage retinal pigment epithelial cells

We examined the gene expression profiles of retinal pigment epithelial (RPE) cells which were aged in vitro by repeated passage. RPE cells from human eyes were cultured to passage 3-5 (early passage) or 19-21 (late passage) and used to study gene expression profiles by cDNA microarray. Results from microarray analysis were further confirmed by real-time PCR. Microarray analysis showed gene expression changes among 588 known genes. The expression levels of 15 genes (2.6%) increased in late passage RPE cells, while 43 genes (7.3%) decreased using a two-fold criterion. These differentially expressed genes encompassed many functional classes. A small number of stress genes, such as clusterin, replication protein A and Ku80, were up-regulated. The down-regulated genes included many enzymes of energy and biomolecule metabolism as well as cell cycle proteins and cell adhesion proteins. Results from real-time PCR were generally consistent with microarray findings. The expression levels of the examined angiogenic factors were either unchanged or down-regulated. Comparing early (p=3-5) and late (p=9-12) passage RPE cells, several categories of differentially expressed genes were identified. However, there was no enhanced expression of known angiogenic factors.     

氧化损伤对体外培养人视网膜色素上皮细胞PEDF的影响

目的研究氧化损伤对人视网膜色素上皮(retinal pigment epithelial,RPE)细胞表达色素上皮细胞衍生因子(pigment epithelium derived factor,PEDF)的影响。方法体外培养人RPE细胞,加入浓度为600μmol.L-1H2O2分别作用不同时间(2h、8h、24h),采用免疫细胞化学法检测PEDF蛋白的表达,逆转录聚合酶链式反应(RT-PCR)法检测PEDF mRNA。结果免疫细胞化学染色对照组即有PEDF的阳性表达,而氧化损伤2h、8h、24h PEDF蛋白阳性表达量逐渐减少,染色由棕黄色变为黄色。各时间段两组结果比较,差异均具有统计学意义(P<0.05)。RT-PCR检测PEDF mRNA量与PEDF蛋白表达变化一致。结论氧化损伤能促使人RPE细胞PEDF表达下调,并在一定范围内与作用时间有关。