中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2013年
11期
1037-1044
,共8页
张丽娟%曲进锋%白玉婧%孙摇遥%于文贞%黄旅珍%黎晓新%赵明威
張麗娟%麯進鋒%白玉婧%孫搖遙%于文貞%黃旅珍%黎曉新%趙明威
장려연%곡진봉%백옥청%손요요%우문정%황려진%려효신%조명위
辅酶Q10%视网膜/色素上皮细胞%氧化应激/损伤%凋亡
輔酶Q10%視網膜/色素上皮細胞%氧化應激/損傷%凋亡
보매Q10%시망막/색소상피세포%양화응격/손상%조망
Coenzyme Q10%Retina/pigment epithelial cell%Oxidative stress/injury%Apoptosis
背景 有多种因素参与年龄相关性黄斑变性(AMD)的发病,其中视网膜色素上皮(RPE)细胞的氧化应激损伤与AMD的发病密切相关.实验和临床研究表明,辅酶Q10是一种强抗氧化剂,探讨其对人RPE细胞的氧化应激损伤是否有保护作用对于AMD的防治有重要的临床意义. 目的 探讨辅酶Q10对体外培养的人RPE细胞氧化应激损伤的保护作用及其作用机制. 方法 体外培养人RPE细胞,分为正常对照组(单纯培养基培养)、不同浓度(0.01、1、100 μmol/L)辅酶Q10预处理组和单纯叔丁基过氧化氢(TBHP)处理组,其中各浓度辅酶Q10预处理组以辅酶Q10预处理后暴露于TBHP,光学显微镜下观察各组RPE细胞的形态；用细胞计数试剂盒-8(CCK-8)法检测各组RPE细胞活性的变化；Annexin V-FITC/PI染色后用流式细胞仪检测细胞凋亡情况；TBARS法检测细胞的脂质过氧化水平；用DCFH-DA荧光法检测细胞内活性氧簇(ROS)水平；实时定量PCR法检测RPE细胞中促凋亡基因Fas mRNA的表达；Western blot法检测细胞中Fas蛋白的表达.结果 单纯TBHP处理组RPE细胞贴壁数量较正常对照组明显减少,而0.01、1、100 μmol/L辅酶Q10预处理组贴壁细胞数较单纯TBHP处理组增加,高浓度辅酶Q10预处理组细胞形态的改善和细胞存活的数目更接近正常对照组.与单纯TBHP处理组比较,1μmol/L、100 μmol/L辅酶Q10预处理组RPE细胞吸光度(A450)值明显增高(0.52±0.10 vs.0.25 ±0.03、0.59±0.06 vs.0.25±0.03),差异均有统计学意义(P=0.041、0.002);RPE细胞的凋亡率明显下降[(72.1±6.6)％vs.(91.7±2.3)％、(69.0±4.4)％ vs.(91.7±2.3)％],差异均有统计学意义(P=0.032、0.004)；0.01、1、100 μmol/L辅酶Q10预处理组的细胞脂质过氧化水平依次降低,与单纯TBHP处理组比较差异均有统计学意义(P=0.047、0.030、0.015),与单纯TBHP组相比,0.01、1、100 μmol/L辅酶Q10预处理组细胞内ROS水平逐渐下降,1μmol/L、100tμmol/L辅酶Q10预处理组细胞内ROS水平明显低于单纯TBHP处理组(5.25±0.90 vs.11.39±2.30、7.91 ±0.80 vs.11.39±2.30),差异均有统计学意义(P=0.028、0.007)；与单纯TBHP处理组相比,辅酶Q10预处理组细胞中Fas mRNA表达量均有不同程度的下降,1 μmol/L、100 μmol/L辅酶Q10预处理组细胞中Fas mRNA表达量的差异均有统计学意义(P=0.049、0.008)；0.01、1、100μmol/L辅酶Q10预处理组细胞中Fas蛋白表达量均明显下降,差异均有统计学意义(P=0.001、0.000、0.000). 结论 辅酶Q10对体外培养的人RPE细胞具有保护作用,其作用在一定范围内呈浓度依赖性,这种保护作用可能与减少细胞的氧化应激损伤和抑制细胞凋亡有关.
揹景 有多種因素參與年齡相關性黃斑變性(AMD)的髮病,其中視網膜色素上皮(RPE)細胞的氧化應激損傷與AMD的髮病密切相關.實驗和臨床研究錶明,輔酶Q10是一種彊抗氧化劑,探討其對人RPE細胞的氧化應激損傷是否有保護作用對于AMD的防治有重要的臨床意義. 目的 探討輔酶Q10對體外培養的人RPE細胞氧化應激損傷的保護作用及其作用機製. 方法 體外培養人RPE細胞,分為正常對照組(單純培養基培養)、不同濃度(0.01、1、100 μmol/L)輔酶Q10預處理組和單純叔丁基過氧化氫(TBHP)處理組,其中各濃度輔酶Q10預處理組以輔酶Q10預處理後暴露于TBHP,光學顯微鏡下觀察各組RPE細胞的形態；用細胞計數試劑盒-8(CCK-8)法檢測各組RPE細胞活性的變化；Annexin V-FITC/PI染色後用流式細胞儀檢測細胞凋亡情況；TBARS法檢測細胞的脂質過氧化水平；用DCFH-DA熒光法檢測細胞內活性氧簇(ROS)水平；實時定量PCR法檢測RPE細胞中促凋亡基因Fas mRNA的錶達；Western blot法檢測細胞中Fas蛋白的錶達.結果 單純TBHP處理組RPE細胞貼壁數量較正常對照組明顯減少,而0.01、1、100 μmol/L輔酶Q10預處理組貼壁細胞數較單純TBHP處理組增加,高濃度輔酶Q10預處理組細胞形態的改善和細胞存活的數目更接近正常對照組.與單純TBHP處理組比較,1μmol/L、100 μmol/L輔酶Q10預處理組RPE細胞吸光度(A450)值明顯增高(0.52±0.10 vs.0.25 ±0.03、0.59±0.06 vs.0.25±0.03),差異均有統計學意義(P=0.041、0.002);RPE細胞的凋亡率明顯下降[(72.1±6.6)％vs.(91.7±2.3)％、(69.0±4.4)％ vs.(91.7±2.3)％],差異均有統計學意義(P=0.032、0.004)；0.01、1、100 μmol/L輔酶Q10預處理組的細胞脂質過氧化水平依次降低,與單純TBHP處理組比較差異均有統計學意義(P=0.047、0.030、0.015),與單純TBHP組相比,0.01、1、100 μmol/L輔酶Q10預處理組細胞內ROS水平逐漸下降,1μmol/L、100tμmol/L輔酶Q10預處理組細胞內ROS水平明顯低于單純TBHP處理組(5.25±0.90 vs.11.39±2.30、7.91 ±0.80 vs.11.39±2.30),差異均有統計學意義(P=0.028、0.007)；與單純TBHP處理組相比,輔酶Q10預處理組細胞中Fas mRNA錶達量均有不同程度的下降,1 μmol/L、100 μmol/L輔酶Q10預處理組細胞中Fas mRNA錶達量的差異均有統計學意義(P=0.049、0.008)；0.01、1、100μmol/L輔酶Q10預處理組細胞中Fas蛋白錶達量均明顯下降,差異均有統計學意義(P=0.001、0.000、0.000). 結論 輔酶Q10對體外培養的人RPE細胞具有保護作用,其作用在一定範圍內呈濃度依賴性,這種保護作用可能與減少細胞的氧化應激損傷和抑製細胞凋亡有關.
배경 유다충인소삼여년령상관성황반변성(AMD)적발병,기중시망막색소상피(RPE)세포적양화응격손상여AMD적발병밀절상관.실험화림상연구표명,보매Q10시일충강항양화제,탐토기대인RPE세포적양화응격손상시부유보호작용대우AMD적방치유중요적림상의의. 목적 탐토보매Q10대체외배양적인RPE세포양화응격손상적보호작용급기작용궤제. 방법 체외배양인RPE세포,분위정상대조조(단순배양기배양)、불동농도(0.01、1、100 μmol/L)보매Q10예처리조화단순숙정기과양화경(TBHP)처리조,기중각농도보매Q10예처리조이보매Q10예처리후폭로우TBHP,광학현미경하관찰각조RPE세포적형태；용세포계수시제합-8(CCK-8)법검측각조RPE세포활성적변화；Annexin V-FITC/PI염색후용류식세포의검측세포조망정황；TBARS법검측세포적지질과양화수평；용DCFH-DA형광법검측세포내활성양족(ROS)수평；실시정량PCR법검측RPE세포중촉조망기인Fas mRNA적표체；Western blot법검측세포중Fas단백적표체.결과 단순TBHP처리조RPE세포첩벽수량교정상대조조명현감소,이0.01、1、100 μmol/L보매Q10예처리조첩벽세포수교단순TBHP처리조증가,고농도보매Q10예처리조세포형태적개선화세포존활적수목경접근정상대조조.여단순TBHP처리조비교,1μmol/L、100 μmol/L보매Q10예처리조RPE세포흡광도(A450)치명현증고(0.52±0.10 vs.0.25 ±0.03、0.59±0.06 vs.0.25±0.03),차이균유통계학의의(P=0.041、0.002);RPE세포적조망솔명현하강[(72.1±6.6)％vs.(91.7±2.3)％、(69.0±4.4)％ vs.(91.7±2.3)％],차이균유통계학의의(P=0.032、0.004)；0.01、1、100 μmol/L보매Q10예처리조적세포지질과양화수평의차강저,여단순TBHP처리조비교차이균유통계학의의(P=0.047、0.030、0.015),여단순TBHP조상비,0.01、1、100 μmol/L보매Q10예처리조세포내ROS수평축점하강,1μmol/L、100tμmol/L보매Q10예처리조세포내ROS수평명현저우단순TBHP처리조(5.25±0.90 vs.11.39±2.30、7.91 ±0.80 vs.11.39±2.30),차이균유통계학의의(P=0.028、0.007)；여단순TBHP처리조상비,보매Q10예처리조세포중Fas mRNA표체량균유불동정도적하강,1 μmol/L、100 μmol/L보매Q10예처리조세포중Fas mRNA표체량적차이균유통계학의의(P=0.049、0.008)；0.01、1、100μmol/L보매Q10예처리조세포중Fas단백표체량균명현하강,차이균유통계학의의(P=0.001、0.000、0.000). 결론 보매Q10대체외배양적인RPE세포구유보호작용,기작용재일정범위내정농도의뢰성,저충보호작용가능여감소세포적양화응격손상화억제세포조망유관.
Background Multiple factors are associated with the pathogenesis of age-related macular degeneration (AMD),and the oxidative stress injury of retinal pigment epithelial (RPE) cells is thought to play a key role.Coenzyme Q10 (CoQ10) is verified to be a powerful antioxidant.Exploring if CoQ10 has a protective effect on RPE cells from oxidative stress injury is of important significance for the prevention and treatment of AMD.Objective This study was conducted to investigate the protective efficacy of CoQ10 on RPE cells against oxidative stress and its potential mechanisms.Methods Human RPE cell line,D407,was used for the study.The RPE cells were subjected to oxidative stress with 400 μmol/L tert-butyl hydroperoxide (TBHP) in the absence or presence of CoQ10 at different concentrations (0.01,1,100 μmol/L).The cell viability was determined using a cell counting kit8 (CCK-8) assay.Apoptosis was detected by flow cytometry(FCM) using annexin V-FITC and PI staining.The levels of oxidative stress in the cells that were exposed to different oxidizing conditions were determined using a Thiobarbituric Acid-reactive Substances (TBARS) Assay Kit.Reactive oxidative species (ROS) of the cells were detected with FCM measurements of peroxide-dependent oxidation of 2'-7'-dichlorofluorescein-diacetate (DCFHDA).Expressions of the pro-apoptotic gene Fas mRNA and Fas protein were assayed by real-time PCR and Western blot analysis,respectively.Results The survival RPE cells were obviously less in the only TBHP treatment group than those in the normal control group.However,the number of the survival cells was gradually increased in the 0.01,1,100 μmol/L CoQ10 groups in comparison with the only TBHP treatment group.Compared with only TBHP treatment group,the proliferative value (A450) was significantly enhanced in the 1 μmol/L CoQ10 group and 100 μmol/L CoQ10 group (0.52±0.10 vs.0.25 ±0.03,0.59±0.06 vs.0.25 ±0.03) (P =0.041,0.002),and the cell apoptosis rate was significantly lower than that of RPE cell([72.1 ±6.6] ％ vs.[91.7±2.3] ％,[69.0±4.4] ％ vs.[91.7±2.3]％) (P=0.032,0.004).The lipid peroxidation levels were gradually declined in the 0.01,1,100 μmol/L CoQ10 groups compared with only TBHP treatment group (P=0.047,0.030,0.015),and the ROS in the cells were lowed in the 0.01,1,100 μmol/L CoQ10 groups,showing significant differences between the 1 μmol CoQ10 group or 100 μmol CoQ10 group and only TBHP treatment group (5.25±0.90 vs.11.39±2.30,7.91±0.80 vs.11.39±2.30)(P=0.028,0.007).Compared with only TBHP treatment group,the expressions of Fas mRNA in the cells were considerably decreased in the 1 μmol/L,100 μ mol/L CoQ10 groups (P=0.049,0.008),and the expressions of Fas protein in the cells were considerably decreased in the 0.01,1,100 μmol/L CoQ10 groups (P =0.001,0.000,0.000).Conclusions CoQ10 appears to has protective effects on RPE cells from oxidative stress damage in vitro in a dose-dependent manner probably by down-regulating the pro-apoptotic protein.
