中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2013年
10期
914-918
,共5页
氧化应激%过氧化氢%晶状体上皮细胞%黏着斑激酶
氧化應激%過氧化氫%晶狀體上皮細胞%黏著斑激酶
양화응격%과양화경%정상체상피세포%점착반격매
Oxidative stress%Hydrogen peroxide%Lens epithelial cell%Focal adhesion kinase
背景 年龄相关性白内障的发生、发展与长期氧化应激导致的细胞凋亡有关.研究表明,过氧化氢(H2O2)处理体外培养的细胞后可激活细胞内的黏着斑激酶(FAK)信号通路,从而影响细胞的增生与凋亡.研究FAK对氧化应激晶状体上皮细胞(LECs)的影响在年龄相关性白内障的预防方面有重要意义. 目的 研究H2O2对LECs中FAK表达的影响,探讨FAK对细胞的调控功能. 方法 用含质量分数10％胎牛血清的低糖DMEM培养液在体积分数5％CO2培养箱中常规培养人LECs株HLECs-B3,不含H2O2的为阴性对照组,在含不同浓度(30、50、70、100、300、500、700、1000 μmol/L)H2O2的培养基作用24 h,采用CCK-8法检测各组细胞的存活率；对细胞进行FITC免疫荧光染色,荧光显微镜下观察各组细胞的形态变化及细胞内FAK的表达分布；观察Hoeehst33258染色的细胞核,评估各组细胞的凋亡情况；采用Western blot法技术半定量检测各组细胞内FAK蛋白的表达量及其磷酸化水平. 结果 对照组,30、50、70、100、300、500、700、1000 μmol/L H2O2组HLECs-B3细胞的存活率分别为(1.00±0.03)％、(1.24±0.03)％、(1.36±0.24)％、(0.93±0.02)％、(1.75±0.19)％、(1.37±0.18)％、(0.64±0.01)％、(0.59±0.11)％、(0.14±0.05)％,各组间细胞存活率的差异有统计学意义(F=95.30,P=0.oo),其中300 μmol/L以下的各H2O2组细胞存活率均明显高于对照组,而＞500 μmol/L的各H2O2组细胞存活率均明显低于对照组,差异均有统计学意义(P＜0.05).不同浓度H2O2刺激24 h后,各组HLECs-B3细胞形态由多边形变为长梭形,并伸出伪足,FAK在细胞中呈绿色荧光,其分布随H2O2浓度的变化而发生改变.1000 μmol/L H2O2作用细胞24 h,可见细胞碎片,细胞发生凋亡.Western blot法检测发现,不同浓度H2O2处理HLECs-B3 24 h后,细胞中FAK的表达量明显不同,总体差异有统计学意义(F=28.08,P=0.00),其中＜300 μmol/L的各H2O2组细胞中FAK的表达量明显高于对照组,而＞500 μmol/L的各H2O2组细胞中FAK的表达量明显低于对照组,差异均有统计学意义(P＜0.05).不同浓度H2O2作用3h时测得的磷酸化FAK (p-FAK)水平明显高于作用30 min时的测量值,差异均有统计学意义(P＜0.05). 结论 H2O2可对LECs的存活和形态产生影响,并伴有FAK表达量及其磷酸化水平的改变,提示FAK参与了H2O2对细胞生物学行为的调控过程.
揹景 年齡相關性白內障的髮生、髮展與長期氧化應激導緻的細胞凋亡有關.研究錶明,過氧化氫(H2O2)處理體外培養的細胞後可激活細胞內的黏著斑激酶(FAK)信號通路,從而影響細胞的增生與凋亡.研究FAK對氧化應激晶狀體上皮細胞(LECs)的影響在年齡相關性白內障的預防方麵有重要意義. 目的 研究H2O2對LECs中FAK錶達的影響,探討FAK對細胞的調控功能. 方法 用含質量分數10％胎牛血清的低糖DMEM培養液在體積分數5％CO2培養箱中常規培養人LECs株HLECs-B3,不含H2O2的為陰性對照組,在含不同濃度(30、50、70、100、300、500、700、1000 μmol/L)H2O2的培養基作用24 h,採用CCK-8法檢測各組細胞的存活率；對細胞進行FITC免疫熒光染色,熒光顯微鏡下觀察各組細胞的形態變化及細胞內FAK的錶達分佈；觀察Hoeehst33258染色的細胞覈,評估各組細胞的凋亡情況；採用Western blot法技術半定量檢測各組細胞內FAK蛋白的錶達量及其燐痠化水平. 結果 對照組,30、50、70、100、300、500、700、1000 μmol/L H2O2組HLECs-B3細胞的存活率分彆為(1.00±0.03)％、(1.24±0.03)％、(1.36±0.24)％、(0.93±0.02)％、(1.75±0.19)％、(1.37±0.18)％、(0.64±0.01)％、(0.59±0.11)％、(0.14±0.05)％,各組間細胞存活率的差異有統計學意義(F=95.30,P=0.oo),其中300 μmol/L以下的各H2O2組細胞存活率均明顯高于對照組,而＞500 μmol/L的各H2O2組細胞存活率均明顯低于對照組,差異均有統計學意義(P＜0.05).不同濃度H2O2刺激24 h後,各組HLECs-B3細胞形態由多邊形變為長梭形,併伸齣偽足,FAK在細胞中呈綠色熒光,其分佈隨H2O2濃度的變化而髮生改變.1000 μmol/L H2O2作用細胞24 h,可見細胞碎片,細胞髮生凋亡.Western blot法檢測髮現,不同濃度H2O2處理HLECs-B3 24 h後,細胞中FAK的錶達量明顯不同,總體差異有統計學意義(F=28.08,P=0.00),其中＜300 μmol/L的各H2O2組細胞中FAK的錶達量明顯高于對照組,而＞500 μmol/L的各H2O2組細胞中FAK的錶達量明顯低于對照組,差異均有統計學意義(P＜0.05).不同濃度H2O2作用3h時測得的燐痠化FAK (p-FAK)水平明顯高于作用30 min時的測量值,差異均有統計學意義(P＜0.05). 結論 H2O2可對LECs的存活和形態產生影響,併伴有FAK錶達量及其燐痠化水平的改變,提示FAK參與瞭H2O2對細胞生物學行為的調控過程.
배경 년령상관성백내장적발생、발전여장기양화응격도치적세포조망유관.연구표명,과양화경(H2O2)처리체외배양적세포후가격활세포내적점착반격매(FAK)신호통로,종이영향세포적증생여조망.연구FAK대양화응격정상체상피세포(LECs)적영향재년령상관성백내장적예방방면유중요의의. 목적 연구H2O2대LECs중FAK표체적영향,탐토FAK대세포적조공공능. 방법 용함질량분수10％태우혈청적저당DMEM배양액재체적분수5％CO2배양상중상규배양인LECs주HLECs-B3,불함H2O2적위음성대조조,재함불동농도(30、50、70、100、300、500、700、1000 μmol/L)H2O2적배양기작용24 h,채용CCK-8법검측각조세포적존활솔；대세포진행FITC면역형광염색,형광현미경하관찰각조세포적형태변화급세포내FAK적표체분포；관찰Hoeehst33258염색적세포핵,평고각조세포적조망정황；채용Western blot법기술반정량검측각조세포내FAK단백적표체량급기린산화수평. 결과 대조조,30、50、70、100、300、500、700、1000 μmol/L H2O2조HLECs-B3세포적존활솔분별위(1.00±0.03)％、(1.24±0.03)％、(1.36±0.24)％、(0.93±0.02)％、(1.75±0.19)％、(1.37±0.18)％、(0.64±0.01)％、(0.59±0.11)％、(0.14±0.05)％,각조간세포존활솔적차이유통계학의의(F=95.30,P=0.oo),기중300 μmol/L이하적각H2O2조세포존활솔균명현고우대조조,이＞500 μmol/L적각H2O2조세포존활솔균명현저우대조조,차이균유통계학의의(P＜0.05).불동농도H2O2자격24 h후,각조HLECs-B3세포형태유다변형변위장사형,병신출위족,FAK재세포중정록색형광,기분포수H2O2농도적변화이발생개변.1000 μmol/L H2O2작용세포24 h,가견세포쇄편,세포발생조망.Western blot법검측발현,불동농도H2O2처리HLECs-B3 24 h후,세포중FAK적표체량명현불동,총체차이유통계학의의(F=28.08,P=0.00),기중＜300 μmol/L적각H2O2조세포중FAK적표체량명현고우대조조,이＞500 μmol/L적각H2O2조세포중FAK적표체량명현저우대조조,차이균유통계학의의(P＜0.05).불동농도H2O2작용3h시측득적린산화FAK (p-FAK)수평명현고우작용30 min시적측량치,차이균유통계학의의(P＜0.05). 결론 H2O2가대LECs적존활화형태산생영향,병반유FAK표체량급기린산화수평적개변,제시FAK삼여료H2O2대세포생물학행위적조공과정.
Background The pathogenesis of age-related cataract is associated with the apoptosis of lens epithelial cells (LECs) caused by oxidative stress.Previous studies showed that intracellular focal adhesion kinase (FAK) pathway can be activated by H2O2 in vitro,which induced apoptosis of cells.To investigate the effect of oxidative on FAK expression in LECs is one of important studies in the prevention of age-related cataract.Objective This study was to investigate the expression and function of FAK in human LECs treated by H2O2.Methods Human LECs strain (HLECs-B3) were cultured in vitro in the low glucose DMEM with 10％ fetal bovine serum.Different concentrations (0,30,50,70,100,300,500,700,1000 μmol/L) of H2O2 were added into the culture medium for 24 hours.The survival rate of the cells was detected by Cell Counting Kit-8 (CCK-8) assay.Cell morphology as well as the expression and distribution of FAK in the cells were observed by immunofluorescent staining under the laser confocal microscope.Apoptosis was observed by hoechst33258 staining,and Western blot assay was used to quantitatively detect the expression and phosphorylation of FAK.Results The survival rate of the cells was (1.00±0.03) ％,(1.24±0.03)％,(1.36±0.24) ％,(0.93±0.02)％,(1.75±0.19)％,(1.37±0.18) ％,(0.64±0.01)％,(0.59±0.11)％,(0.14±0.05)％ in 0,30,50,70,100,300,500,700,1000 μmol/L H2O2 groups,with a significant difference among them (F =95.30,P =0.00).The survival rates of the cells in the below 300 μmol/L H2O2 groups were significantly higher than those in the 0 μmol/L H2O2 group,and survival rates of the cells in the above 500 μmol/L H2O2 groups were significantly lower than those in the 0 μmol/L H2O2 group(all at P＜0.05).After H2 O2 treatment for 24 hours,HLECs-B3 cells transformed from polygon shape to spindle shape and extended pseudopodiums,meanwhile the green fluorescence for FAK exhibited in the cytoplasm.Cell apoptosis was found in the 1000 μ mol/L H2O2 group.Western blot assay revealed that the expressing levels (grey scale) were significantly different among the various groups (F=28.08,P=0.00),and FAK expressing levels in the below 300 μmol/L H2O2 groups were significantly higher than those of the 0 μmol/L H2O2 group; while the expressing levels in the above 500 μmol/L H2O2 groups were lower than those of the control 0 μmol/L H202 group (all at P＜0.05).After treated by different concentrations of H2O2,the phosphorylation level of intracellular FAK (p-FAK) was significantly higher in 3 hours group than that in 30 minutes group (all at P＜0.05).Conclusions H2 O2 can affect the survival,proliferation and morphology of human LECs by activating the intracellular FAK pathway,indicating that FAK may play roles in the regulation process of cell biological behavior.
