| | | | | 蓝光致人视网膜色素上皮细胞凋亡与线粒体膜电位和细胞色素C的关系 | | | 蔡善君;严密;毛咏秋;周焰;刘关键 | | | 目的探讨蓝光光照对体外培养的人视网膜色素上皮(RPE)细胞凋亡及对线粒体膜通透性转运功能的影响。方法用蓝光光照体外培养的人RPE细胞。实验分为三部分。第一部分:不同光照度,分为3组,第1组光照度(500±100)lx,第2组光照度(2000±500)lx,第3组光照度(3000±500)lx;光照RPE细胞时间6h,光照结束后24h终止培养。第二部分:同一光照度、不同光照时间,检测RPE细胞亚型时分为3组,第1组光照时间6h,第2组光照时间12h,第3组光照时间24h,光照度均为(2000±500)lx;检测线粒体膜电位时也分为3组,第1组光照时间3h,第2组光照时间6h,第3组光照时间12h,光照度均为(2000±500)lx。第三部分:同一光照度和光照时间,光照度(2000±500)lx,光照RPE细胞时间6h;不同细胞培养终止时间分为4组,第1组终止细胞培养时间为光照后6h,第2组为光照后12h,第3组为光照后24h,第4组为光照后36h。利用末端脱氧核糖核酸转移酶介导的原位缺口末端标记(TUNEL)染色、荧光素标记的AnnexinV-FITC和PI双染流式细胞检测、透射电镜等手段观察RPE细胞凋亡情况。罗丹明123荧光染料孵育人RPE细胞,流式细胞仪检测线粒体膜电位,酶联免疫法测定细胞色素C活性,比色测定法测定Caspase-3的活性。结果第二部分第1组TUNEL染色阳性细胞的体积缩小变圆,胞核浓缩,边聚成新月形或帽状,细胞核碎裂成数个,细胞膜出胞。透射电镜观察发现细胞内线粒体肿胀,线粒体内膜嵴消失,粗面内质网扩张,溶酶体增加。第一部分(500±100)lx光照未引起明显的RPE细胞损伤,但线粒体膜电位明显下降;随着光照度增加,RPE细胞出现凋亡、凋亡继发性坏死及坏死,同时线粒体膜电位逐渐下降。第二部分光照6和12h,凋亡细胞百分比增加,荧光强度明显下降;光照24h凋亡继发性坏死细胞、坏死细胞百分比增加。第三部分光照后6和12h,RPE细胞凋亡百分比逐渐增加;光照后24、36h凋亡继发性坏死细胞和坏死细胞百分比明显增加,光照后各时间点RPE细胞荧光强度明显下降。以光照度(2000±500)lx照射6和12h,可见光照后24和36h两组的细胞色素C浓度明显升高,未检测到Caspase-3活性变化。结论蓝光照射可导致体外培养的人RPE细胞凋亡、凋亡继发性坏死及坏死,其损伤程度与光照度和时间相关;蓝光照射导致培养的人RPE细胞损伤过程与线粒体膜电位降低、细胞色素C释放有关;线粒体膜电位可作为检测蓝光致人RPE细胞凋亡的早期指标。
【作者单位】：遵义医学院附属医院眼科;四川大学华西医院眼科;四川大学肿瘤生物治疗中心实验室;遵义医学院预防医学教研室;四川大学华西临床医学院临床流行病学教研室
【关键词】：光;色素上皮,眼;细胞凋亡;线粒体;膜电位;细胞色素C类
【基金】：国家自然科学基金资助项目(39770788);贵州省科委基金资助项目(3039)
【分类号】：R779.1
【DOI】：CNKI:ISSN:0412-4081.0.2006-12-016
【正文快照】：
视网膜光损伤的病理过程与年龄相关性黄斑变性、视网膜色素变性有许多相似之处,视网膜光损伤是研究视网膜变性类疾病的良好动物模型[1],对其深入的研究可以为这些疾病的临床治疗提供参考依据。在细胞凋亡过程中,线粒体起中心调控作用,就某种意义而言,线粒体可以决定细胞的生存或死亡[2,3]。目前研究者认为[4],线粒体膜通透性转运(mitochondrial permeability transition,MPT)和MPT孔的开放是引起细胞凋亡的直接原因。我们于2003年开始探讨蓝光引起体外培养的人视网膜色素上皮(retinal pigment epithelium,RPE)细胞损伤及其对MPT的影响,… | | |  推荐 CAJ下载  PDF下载 | | | CAJViewer7.0阅读器支持所有CNKI文件格式，AdobeReader仅支持PDF格式 | | | | Relationship between blue light-induced apoptosis and mitochondrial membrane potential and cytochrome C in cultured human retinal pigment epithelium cells | | | CAI Shan-jun;YAN Mi;MAO Yong-qiu;ZHOU Yan;LIU Guan-jian. Department of Ophthalmology;the Affiliated Hospital of Zunyi Medical College;Zunyi 563003;China | | | Objective To investigate the effect of blue light on apoptosis and mitochondrial permeability transition (MPT) of cultured human retinal pigment epithelium (RPE) cells in vitro. Methods Human RPE cells were exposed to blue light (wave length 470-490 nm). The present study consisted of three parts. Part one studied the effect of various intensities of blue light on the RPE cells. Cells were irradiated with (500±100)lx (group 1), (2000±500)lx (group 2) and (3000±500)lx (group 3) blue light, and followed by 24 hours observation. Part two studied the effect of various duration of blue light at identical intensity on the RPE cells. For the study on various subtypes of RPE cells, cells were irradiated by blue light at (2000±500)lx for 6, 12, and 24 hours. For the study of mitochondrial membrane potential, cells were irradiated for 3, 6, and 12 hours. Part three studied cells irradiated with blue light at identical intensity and duration, but with various prolongation of post-exposure culture. The prolongation of post-exposure culture was 6, 12, 24, and 36 hours. Phototoxicity was quantified at various periods after exposure by staining of the nuclei of membrane-compromised cells, by TdT-dUTP terminal nick-end labeling(TUNEL)of apoptotic cells and by Annexin V labeling for phosphatidylserine exposure. Transmission electronmicroscopy was used to determine the ultrastructure changes of RPE cells. Mitochondrial membrane potential (ΔΨ_m) was measured by rhodamine 123 staining and subsequent flow cytometry. Cytochrome C activity was assayed by ELISA. Caspase-3 was detected by colorimetric assay. Results TUNEL-positive labeling cells in first group of part two study showed cell shrinkage, membrane blebbing, apoptotic body, condensation and fragmentation of chromatin. Mitochondrial swelling,extinction of inner mitochondrial membrane ridge, dilation of rough endoplasmic reticulum and increase of the lysosome were also observed in transmission electronmicroscopy. Blue light at (500±100)lx intensity did not induce damage to RPE cells, but decrease of ΔΨ_m was observed. A significant increase of apoptotic,apoptotic necrotic and necrotic percentages, as well as significant decrease of ΔΨ_m were observed at higher light intensity in part one study. Increase of apoptotic percentage was the main response to shorter exposure of blue light. Increase of apoptotic necrotic and necrotic percentage and pronounced decrease of ΔΨ_m occurred in cells irradiated by longer exposure in part two study. In part 3 study, apoptotic response was increased gradually during 6 and 12 hours prolongation of post-exposure culture, more apoptotic necrosis or necrosis were found after post-exposure 24 hours. Decrease of ΔΨ_m was observed in 6 hours prolongation of post-exposure culture and lasting for 48 hours. The concentration of cytochrome C was significantly increased in post-exposure 24 and 36 hours, without any changes of Caspase-3 activity. Conclusions Blue light exposure can induce damages to human RPE cells in vitro, which include apoptosis, apoptotic necrosis and necrosis. These changes are caused by triggering the mitochondrial permeability transition, which results in decrease of ΔΨ_m and release of cytochrome C. ΔΨ_m can be used as a earlier parameter of blue light-induced apoptosis.
【Keyword】：Light;Pigment epithelium of eye;Apoptosis;Mitochondria;Membrane potentials;Cytochrome C |
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