背景 视网膜色素上皮( RPE)细胞的衰老直接影响视网膜光感受器细胞的代谢功能,导致视网膜功能的异常和视力丧失.深入研究RPE细胞的衰老机制将有助于研究年龄相关性黄斑变性(AMD)的预防和治疗. 目的 研究人胚胎RPE细胞在传代过程中的衰老并探讨其机制. 方法 收集胎龄16 ～ 28周的人胎儿眼球6个,对人胚胎RPE细胞进行分离、培养和传代以制作RPE细胞复制性衰老模型,选择3～12代的RPE细胞用于实验.对培养和传代的RPE细胞用免疫组织化学法以人角蛋白对细胞进行鉴定.应用四甲基偶氮唑蓝(MTT)比色法检测各代细胞的增生活力,以RPE细胞的吸光度(A490)表示.采用流式细胞仪检测传代细胞周期和细胞线粒体跨膜电位(△ψm)的变化.结果 培养和传代的细胞呈六角形,人角蛋白检测呈棕色,第3代以后的细胞色素消失,3～6代的细胞形态正常,生长活跃,之后生长能力和增生活力下降.培养第3 ～12代RPE细胞随着传代的增加,处于G0/G1期的细胞百分比逐渐增加,差异有统计学意义(F=180.430,P=0.000),由第3代细胞的68.40％增加到第12代细胞的87.33％,细胞生长趋于停滞.各代间处于G0/G1期细胞的百分比比较,第6代细胞明显高于第3代,第9代明显高于第6代,第12代则明显高于第9代,差异均有统计学意义(t=4.002,P＜0.05；t=12.885,P＜0.01；t=16.387,P＜0.01).MTT法对RPE细胞活力的检测结果表明,随着传代次数的增多,反映RPE细胞活力的A490值逐渐下降,总体比较差异有统计学意义(F=38.770,P=0.000),第9代、第12代RPE细胞的A490值分别明显低于第6代和第9代,差异均有统计学意义(t=5.991、11.983,P＜0.01).同时,3～12代PRE细胞随着传代的增加,罗丹明123染色荧光强度逐渐降低,差异有统计学意义(F=121.680,P=0.000),第6、9、12代细胞的平均荧光强度分别低于第3、6、9代,差异均有统计学意义(t=6.918、7.620、11.207,P＜0.01). 结论 成功制作了RPE细胞随年龄而逐渐衰老的模型,提示衰老细胞线粒体膜电位降低,线粒体功能受损可能是RPE细胞衰老的机制之一.
揹景 視網膜色素上皮( RPE)細胞的衰老直接影響視網膜光感受器細胞的代謝功能,導緻視網膜功能的異常和視力喪失.深入研究RPE細胞的衰老機製將有助于研究年齡相關性黃斑變性(AMD)的預防和治療. 目的 研究人胚胎RPE細胞在傳代過程中的衰老併探討其機製. 方法 收集胎齡16 ～ 28週的人胎兒眼毬6箇,對人胚胎RPE細胞進行分離、培養和傳代以製作RPE細胞複製性衰老模型,選擇3～12代的RPE細胞用于實驗.對培養和傳代的RPE細胞用免疫組織化學法以人角蛋白對細胞進行鑒定.應用四甲基偶氮唑藍(MTT)比色法檢測各代細胞的增生活力,以RPE細胞的吸光度(A490)錶示.採用流式細胞儀檢測傳代細胞週期和細胞線粒體跨膜電位(△ψm)的變化.結果 培養和傳代的細胞呈六角形,人角蛋白檢測呈棕色,第3代以後的細胞色素消失,3～6代的細胞形態正常,生長活躍,之後生長能力和增生活力下降.培養第3 ～12代RPE細胞隨著傳代的增加,處于G0/G1期的細胞百分比逐漸增加,差異有統計學意義(F=180.430,P=0.000),由第3代細胞的68.40％增加到第12代細胞的87.33％,細胞生長趨于停滯.各代間處于G0/G1期細胞的百分比比較,第6代細胞明顯高于第3代,第9代明顯高于第6代,第12代則明顯高于第9代,差異均有統計學意義(t=4.002,P＜0.05；t=12.885,P＜0.01；t=16.387,P＜0.01).MTT法對RPE細胞活力的檢測結果錶明,隨著傳代次數的增多,反映RPE細胞活力的A490值逐漸下降,總體比較差異有統計學意義(F=38.770,P=0.000),第9代、第12代RPE細胞的A490值分彆明顯低于第6代和第9代,差異均有統計學意義(t=5.991、11.983,P＜0.01).同時,3～12代PRE細胞隨著傳代的增加,囉丹明123染色熒光彊度逐漸降低,差異有統計學意義(F=121.680,P=0.000),第6、9、12代細胞的平均熒光彊度分彆低于第3、6、9代,差異均有統計學意義(t=6.918、7.620、11.207,P＜0.01). 結論 成功製作瞭RPE細胞隨年齡而逐漸衰老的模型,提示衰老細胞線粒體膜電位降低,線粒體功能受損可能是RPE細胞衰老的機製之一.
배경 시망막색소상피( RPE)세포적쇠로직접영향시망막광감수기세포적대사공능,도치시망막공능적이상화시력상실.심입연구RPE세포적쇠로궤제장유조우연구년령상관성황반변성(AMD)적예방화치료. 목적 연구인배태RPE세포재전대과정중적쇠로병탐토기궤제. 방법 수집태령16 ～ 28주적인태인안구6개,대인배태RPE세포진행분리、배양화전대이제작RPE세포복제성쇠로모형,선택3～12대적RPE세포용우실험.대배양화전대적RPE세포용면역조직화학법이인각단백대세포진행감정.응용사갑기우담서람(MTT)비색법검측각대세포적증생활력,이RPE세포적흡광도(A490)표시.채용류식세포의검측전대세포주기화세포선립체과막전위(△ψm)적변화.결과 배양화전대적세포정륙각형,인각단백검측정종색,제3대이후적세포색소소실,3～6대적세포형태정상,생장활약,지후생장능력화증생활력하강.배양제3 ～12대RPE세포수착전대적증가,처우G0/G1기적세포백분비축점증가,차이유통계학의의(F=180.430,P=0.000),유제3대세포적68.40％증가도제12대세포적87.33％,세포생장추우정체.각대간처우G0/G1기세포적백분비비교,제6대세포명현고우제3대,제9대명현고우제6대,제12대칙명현고우제9대,차이균유통계학의의(t=4.002,P＜0.05；t=12.885,P＜0.01；t=16.387,P＜0.01).MTT법대RPE세포활력적검측결과표명,수착전대차수적증다,반영RPE세포활력적A490치축점하강,총체비교차이유통계학의의(F=38.770,P=0.000),제9대、제12대RPE세포적A490치분별명현저우제6대화제9대,차이균유통계학의의(t=5.991、11.983,P＜0.01).동시,3～12대PRE세포수착전대적증가,라단명123염색형광강도축점강저,차이유통계학의의(F=121.680,P=0.000),제6、9、12대세포적평균형광강도분별저우제3、6、9대,차이균유통계학의의(t=6.918、7.620、11.207,P＜0.01). 결론 성공제작료RPE세포수년령이축점쇠로적모형,제시쇠로세포선립체막전위강저,선립체공능수손가능시RPE세포쇠로적궤제지일.
Background Retinal pigment epithelial (RPE) cell senescence damage the metabolism of photoreceptor,leading to retinal dysfunction and loss of vision.To understand RPE cell senescence mechanism will contribute to the study of age-related macular degeneration ( AMD). Objective The present study was to prepare the ageing RPE cell model with passage and explore its potential mechanisms. Methods This study was approved by the Ethic Committee of Qingdao University Medical College,and the informed consent was obtained from each gravida.Six human eyeballs were obtained from artificial labor fetusl with the gestational age 16-28 weeks.RPE cells were isolated,cultured and passaged in vitro to establish the cell replicative aging model.The third to twelfth cells were collected to be used to this experiment.Human keratin was used to identify the cells by immunochemistry,and MTT method was utilized to assess the proliferation and viability of different generations of cells as the A490 value.The cellular cycles and transmembrane potential (△ψm) of mitochondrion (△ψm) with passage were detected and compared using Flow Cytometry. Results Cultured and passaged cells showed the hexagon in shape with the melanin in 1-2 generations of cells and presented with the brown staining in cytoplasm for human keratin.The melanin was absent in the third generation cells.Vibrant growth statues were seen from the 3-6 generations cells and thereafter the proliferation ability reduced.The cells of G0/G1 phase were gradually elevated with the passage from 3 - 12 generations with a percentage of 68.40％ in the third generation of cells to 87.33％ in the twelfth generation of cells,showing a significant difference among various generations ( F =180.43,P =0.00),and that of the sixth,ninth and twelfth generation of cells was significant higher than the third,sixth and ninth generation respectively (t =4.002,P＜0.05 ; t=12.885,P＜0.01 ;t=16.387,P＜0.01 ).MTT assay showed that of RPE cells were significantly declined with the passage ( F =38.77,P =0.00),and the A490 value of the ninth,twelfth generations of cells was considerably lower in comparison with sixth and ninth generation respectively ( t =5.991,11.983,P＜0.01 ).From 3 through 12 generations of cells,the staining intensity of rhodamine 123 was gradually decreased ( F =121.68,P =0.00 ),and the staining intensity in the sixth,ninth and twelfth generation of cells was significant lower than that the third,sixth and ninth generation respectively(t=6.918,7.620,11.207,P＜0.01 ). Conclusions A replicative aging model can be successfully created by the passage in vitro using human fetal RPE cells.The reduce of transmembrane potential and damage of mitochondria might be one of mechanisms of senescence of RPE cells.
