中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2015年
2期
109-114
,共6页
边江%曲明俐%王瑶%杨玲玲%史伟云%周庆军
邊江%麯明俐%王瑤%楊玲玲%史偉雲%週慶軍
변강%곡명리%왕요%양령령%사위운%주경군
圆锥角膜%核因子E2相关因子2-抗氧化反应元件信号通路%角膜基质细胞%氧化应激
圓錐角膜%覈因子E2相關因子2-抗氧化反應元件信號通路%角膜基質細胞%氧化應激
원추각막%핵인자E2상관인자2-항양화반응원건신호통로%각막기질세포%양화응격
Keratoconus%Nrf2-ARE signaling%Corneal stromal cell%Oxidative stress
背景 氧化应激在圆锥角膜发病过程中具有重要的作用,核因子E2相关因子2-抗氧化反应元件(Nrf2-ARE)信号通路是介导细胞氧化应激反应的关键通路,但其在圆锥角膜发病中的作用及其机制鲜见报道. 目的 研究正常角膜与圆锥角膜基质细胞中Nrf2-ARE信号通路活化的区别及Nrf2-ARE通路对角膜基质降解酶表达水平的影响,探讨圆锥角膜发病的具体机制.方法 于2012年11月至2013年6月在青岛眼科医院收集圆锥角膜患者术中的角膜组织样本和正常供体角膜样本,采用中性蛋白酶和胶原蛋白酶联合消化法分离角膜基质细胞并用含质量分数10％胎牛血清的DMEM/F12培养基培养细胞,待细胞80％融合后在培养基中加入200 μmol/L H2O2处理1h以模拟氧化应激微环境.采用DCFH-DA荧光底物孵育法检测细胞内活性氧簇(ROS)含量,分别采用Western blot和实时定量PCR法检测细胞核内Nrf2mRNA及其蛋白、Nrf2-ARE信号通路下游抗氧化蛋白、尿激酶型纤溶酶原激活物(uPA)、uPA受体(uPAR) mRNA及其蛋白的相对表达水平,采用明胶酶谱法检测细胞中基质金属蛋白酶2(MMP-2)活性.结果 正常培养条件下,圆锥角膜基质细胞中ROS荧光强于正常角膜基质细胞,细胞核内Nrf2蛋白表达水平均明显高于正常角膜基质细胞,差异有统计学意义(t=18.155,P＜0.01),但在H2O2处理条件下,圆锥角膜基质细胞中ROS荧光强度明显强于正常角膜基质细胞,且圆锥角膜基质细胞核内Nrf2表达水平明显低于正常培养条件下的圆锥角膜基质细胞,差异有统计学意义(t=62.123,P＜0.01).正常培养条件下,圆锥角膜基质细胞间还原型烟酰胺腺嘌呤二核苷酸磷酸氧化还原酶1(NQO-1)、血红素氧合酶1(HO-1)、超氧化物歧化酶2(SOD2) mRNA及其蛋白的相对表达量明显低于正常角膜基质细胞,差异均有统计学意义(均P＜0.01);但在H2O2培养条件下2种细胞间未见明显变化(NQO-1:t=2.209,P=0.092;HO-1:t=0.293,P=0.784;SOD2:t=0.749,P=0.495);圆锥角膜基质细胞uPA、uPAR表达量和MMP-2活性均明显高于正常角膜基质细胞,差异均有统计学意义(t=19.164、15.458、4.818,均P＜0.01). 结论 圆锥角膜基质细胞在Nrf2-ARE信号通路活化方面存在缺陷,且这种缺陷与其表达基质降解酶的水平密切相关,说明该通路异常可能是圆锥角膜发病的机制之一.
揹景 氧化應激在圓錐角膜髮病過程中具有重要的作用,覈因子E2相關因子2-抗氧化反應元件(Nrf2-ARE)信號通路是介導細胞氧化應激反應的關鍵通路,但其在圓錐角膜髮病中的作用及其機製鮮見報道. 目的 研究正常角膜與圓錐角膜基質細胞中Nrf2-ARE信號通路活化的區彆及Nrf2-ARE通路對角膜基質降解酶錶達水平的影響,探討圓錐角膜髮病的具體機製.方法 于2012年11月至2013年6月在青島眼科醫院收集圓錐角膜患者術中的角膜組織樣本和正常供體角膜樣本,採用中性蛋白酶和膠原蛋白酶聯閤消化法分離角膜基質細胞併用含質量分數10％胎牛血清的DMEM/F12培養基培養細胞,待細胞80％融閤後在培養基中加入200 μmol/L H2O2處理1h以模擬氧化應激微環境.採用DCFH-DA熒光底物孵育法檢測細胞內活性氧簇(ROS)含量,分彆採用Western blot和實時定量PCR法檢測細胞覈內Nrf2mRNA及其蛋白、Nrf2-ARE信號通路下遊抗氧化蛋白、尿激酶型纖溶酶原激活物(uPA)、uPA受體(uPAR) mRNA及其蛋白的相對錶達水平,採用明膠酶譜法檢測細胞中基質金屬蛋白酶2(MMP-2)活性.結果 正常培養條件下,圓錐角膜基質細胞中ROS熒光彊于正常角膜基質細胞,細胞覈內Nrf2蛋白錶達水平均明顯高于正常角膜基質細胞,差異有統計學意義(t=18.155,P＜0.01),但在H2O2處理條件下,圓錐角膜基質細胞中ROS熒光彊度明顯彊于正常角膜基質細胞,且圓錐角膜基質細胞覈內Nrf2錶達水平明顯低于正常培養條件下的圓錐角膜基質細胞,差異有統計學意義(t=62.123,P＜0.01).正常培養條件下,圓錐角膜基質細胞間還原型煙酰胺腺嘌呤二覈苷痠燐痠氧化還原酶1(NQO-1)、血紅素氧閤酶1(HO-1)、超氧化物歧化酶2(SOD2) mRNA及其蛋白的相對錶達量明顯低于正常角膜基質細胞,差異均有統計學意義(均P＜0.01);但在H2O2培養條件下2種細胞間未見明顯變化(NQO-1:t=2.209,P=0.092;HO-1:t=0.293,P=0.784;SOD2:t=0.749,P=0.495);圓錐角膜基質細胞uPA、uPAR錶達量和MMP-2活性均明顯高于正常角膜基質細胞,差異均有統計學意義(t=19.164、15.458、4.818,均P＜0.01). 結論 圓錐角膜基質細胞在Nrf2-ARE信號通路活化方麵存在缺陷,且這種缺陷與其錶達基質降解酶的水平密切相關,說明該通路異常可能是圓錐角膜髮病的機製之一.
배경 양화응격재원추각막발병과정중구유중요적작용,핵인자E2상관인자2-항양화반응원건(Nrf2-ARE)신호통로시개도세포양화응격반응적관건통로,단기재원추각막발병중적작용급기궤제선견보도. 목적 연구정상각막여원추각막기질세포중Nrf2-ARE신호통로활화적구별급Nrf2-ARE통로대각막기질강해매표체수평적영향,탐토원추각막발병적구체궤제.방법 우2012년11월지2013년6월재청도안과의원수집원추각막환자술중적각막조직양본화정상공체각막양본,채용중성단백매화효원단백매연합소화법분리각막기질세포병용함질량분수10％태우혈청적DMEM/F12배양기배양세포,대세포80％융합후재배양기중가입200 μmol/L H2O2처리1h이모의양화응격미배경.채용DCFH-DA형광저물부육법검측세포내활성양족(ROS)함량,분별채용Western blot화실시정량PCR법검측세포핵내Nrf2mRNA급기단백、Nrf2-ARE신호통로하유항양화단백、뇨격매형섬용매원격활물(uPA)、uPA수체(uPAR) mRNA급기단백적상대표체수평,채용명효매보법검측세포중기질금속단백매2(MMP-2)활성.결과 정상배양조건하,원추각막기질세포중ROS형광강우정상각막기질세포,세포핵내Nrf2단백표체수평균명현고우정상각막기질세포,차이유통계학의의(t=18.155,P＜0.01),단재H2O2처리조건하,원추각막기질세포중ROS형광강도명현강우정상각막기질세포,차원추각막기질세포핵내Nrf2표체수평명현저우정상배양조건하적원추각막기질세포,차이유통계학의의(t=62.123,P＜0.01).정상배양조건하,원추각막기질세포간환원형연선알선표령이핵감산린산양화환원매1(NQO-1)、혈홍소양합매1(HO-1)、초양화물기화매2(SOD2) mRNA급기단백적상대표체량명현저우정상각막기질세포,차이균유통계학의의(균P＜0.01);단재H2O2배양조건하2충세포간미견명현변화(NQO-1:t=2.209,P=0.092;HO-1:t=0.293,P=0.784;SOD2:t=0.749,P=0.495);원추각막기질세포uPA、uPAR표체량화MMP-2활성균명현고우정상각막기질세포,차이균유통계학의의(t=19.164、15.458、4.818,균P＜0.01). 결론 원추각막기질세포재Nrf2-ARE신호통로활화방면존재결함,차저충결함여기표체기질강해매적수평밀절상관,설명해통로이상가능시원추각막발병적궤제지일.
Background Recent researches show that oxidative stress is involved in the progress of keratoconus.Nuclear factor-E2-related factor 2-antioxidant response element (Nrf2-ARE) pathway plays a critical role in the defense against oxidative stress,but its function in keratoconus is unclear.Objective To investigate the differences of Nrf2-ARE signaling activation and matrix degenerating enzymes between keratoconus and normal corneal stromal cells.Methods Corneal stromal cells were isolated from keratoconus and normal cornea by using dispase and collagenase digestion.The cells were treated with hydrogen peroxide (H2O2) to mimic in vivo oxidative stress condition.Reactive oxygen species (ROS) production was measured by fluorescence substrate DCHF-DA incubation.Nrf2 level and the expression of Nrf2-ARE downstream antioxidant genes were analyzed by Western blot and real-time quantitative-PCR(RT-qPCR).The activity of matrix degenerating enzymes,including urokinase-type plasminogen activator (uPA)-uPA receptor (uPAR) system and matrix metalloproteinase-2 (MMP-2) were assessed by Western blot and gelatin zymography respectively.Results In normal culture,keratoconus corneal stromal cells assumed increased basal ROS and Nrf2 level when compared with normal cells(t =18.155,P＜0.01).However,after H2O2 treatment,the keratoconus corneal stromal cells showed increased ROS production,while decreased Nrf2 translocation and no significant difference in expression levels of Nrf2-ARE downstream antioxidant genes (Nrf2:t =62.123,P＜ 0.01 ; (nicotinamide adenine dinucleotide phosphate quinine oxidoreductase-1 [NQO-1]:t =2.209,P =0.092 ; hemo oxygenase-1 [HO-1]:t =0.293,P =0.784 ; superoxide dismutase [SOD2]:t =0.749,P =0.495).The contents of uPA-uPAR and the activity of MMP-2 also showed a higher level in keratoconus corneal stromal cells than normal cells,with significant differences between them (t =19.164,15.458,4.818,all at P＜0.01).Conclusions The defect of Nrf2-ARE signaling activation exists in the keratoconus corneal stromal cells,and correlats with the abnormal expression level of stromal degeneration enzymes,which suggests that the defect of Nrf2-ARE signaling activation may be involved in the progression of keratoconus.
