中华眼科杂志
中華眼科雜誌
중화안과잡지
Chinese Journal of Ophthalmology
2013年
8期
716-722
,共7页
李轩%姜志昕%郝朋%汤欣
李軒%薑誌昕%郝朋%湯訢
리헌%강지흔%학붕%탕흔
上皮,角膜%上皮细胞%胸腺素%氧化性应激%细胞凋亡%细胞,培养的
上皮,角膜%上皮細胞%胸腺素%氧化性應激%細胞凋亡%細胞,培養的
상피,각막%상피세포%흉선소%양화성응격%세포조망%세포,배양적
Epithelium,corneal%Epithelial cells%Thymosin%Oxidative stress%Apoptosis%Cells,cultured
目的 探讨胸腺素β4(Tβ4)对体外培养的兔角膜上皮细胞氧化损伤的影响.方法 实验研究.采用组织块培养法获得原代兔角膜上皮细胞并进行传代培养.采用形态学观察和逆转录聚合酶链反应(RT-PCR)法检测成熟角蛋白(keratin) 12和缝隙连接蛋白(connexin) 43的表达情况,对细胞进行鉴定.以H2O2处理角膜上皮细胞制备体外细胞氧化损伤模型,并观测Tβ4对其保护作用.实验分为4组:正常对照组、Tβ4组、H2O2组和H2O2+ Tβ4组.采用MTT比色法检测角膜上皮细胞存活度；运用TUNEL染色观察角膜上皮细胞凋亡；利用DCFH-DA荧光探针检测细胞内活性氧(ROS)水平；通过划痕实验观察角膜上皮细胞的迁移运动能力.组间均数比较采用方差分析,进一步两两比较采用LSD-t检验.结果 培养的细胞呈圆形、卵圆形和多边形,铺路石样生长；RT-PCR法检测显示培养的细胞表达角膜上皮细胞特异性基因,keratin 12和connexin 43,呈现角膜上皮细胞的特征.经H2O2刺激后,角膜上皮细胞的存活率(67.20％±5.87％)较正常对照组显著下降(l00.00％±9.99％)(4个组间比较F=18.61,P=0.001,两两比较P=0.000)；H2O2+Tβ4组细胞存活率(83.42％±7.43％)与H2O2组(67.20％±5.87％)比较明显增高(P =0.023).4个组间细胞划痕实验结果的差异具有统计学意义(F =36.38,P=0.000),在12 hTβ4组划痕已基本消失,细胞迁移率为117.6％ ±2.22％,与正常对照组(100.00％±4.06％)相比显著增加(P=0.005)；12h时H2O2+Tβ4组与H2O2组相比,细胞迁移率为96.57％±8.22％,与H2O2组(64.38％±11.08％)相比明显增加(P=0.000).H2 O2刺激后细胞内ROS水平为234.42％±22.15％,较正常对照组(100.00％±5.28％)明显增加(P =0.000),H2O2+Tβ4组细胞内ROS水平(163.26％±10.53％)虽然较正常对照组偏高,但与H2O2组(234.42％ ±22.15％)比较显著降低(P=0.000).TUNEL染色结果显示,H2O2组呈现较多凋亡细胞,H2 O2+ Tβ4组与H2O2组相比,凋亡细胞明显减少.结论 Tβ4通过抑制氧化损伤后细胞内ROS的产生对氧化应激损伤后的角膜上皮细胞具有减少细胞凋亡,增加细胞存活率并促进细胞迁移的作用,提示Tβ4对角膜上皮细胞氧化损伤具有保护作用.
目的 探討胸腺素β4(Tβ4)對體外培養的兔角膜上皮細胞氧化損傷的影響.方法 實驗研究.採用組織塊培養法穫得原代兔角膜上皮細胞併進行傳代培養.採用形態學觀察和逆轉錄聚閤酶鏈反應(RT-PCR)法檢測成熟角蛋白(keratin) 12和縫隙連接蛋白(connexin) 43的錶達情況,對細胞進行鑒定.以H2O2處理角膜上皮細胞製備體外細胞氧化損傷模型,併觀測Tβ4對其保護作用.實驗分為4組:正常對照組、Tβ4組、H2O2組和H2O2+ Tβ4組.採用MTT比色法檢測角膜上皮細胞存活度；運用TUNEL染色觀察角膜上皮細胞凋亡；利用DCFH-DA熒光探針檢測細胞內活性氧(ROS)水平；通過劃痕實驗觀察角膜上皮細胞的遷移運動能力.組間均數比較採用方差分析,進一步兩兩比較採用LSD-t檢驗.結果 培養的細胞呈圓形、卵圓形和多邊形,鋪路石樣生長；RT-PCR法檢測顯示培養的細胞錶達角膜上皮細胞特異性基因,keratin 12和connexin 43,呈現角膜上皮細胞的特徵.經H2O2刺激後,角膜上皮細胞的存活率(67.20％±5.87％)較正常對照組顯著下降(l00.00％±9.99％)(4箇組間比較F=18.61,P=0.001,兩兩比較P=0.000)；H2O2+Tβ4組細胞存活率(83.42％±7.43％)與H2O2組(67.20％±5.87％)比較明顯增高(P =0.023).4箇組間細胞劃痕實驗結果的差異具有統計學意義(F =36.38,P=0.000),在12 hTβ4組劃痕已基本消失,細胞遷移率為117.6％ ±2.22％,與正常對照組(100.00％±4.06％)相比顯著增加(P=0.005)；12h時H2O2+Tβ4組與H2O2組相比,細胞遷移率為96.57％±8.22％,與H2O2組(64.38％±11.08％)相比明顯增加(P=0.000).H2 O2刺激後細胞內ROS水平為234.42％±22.15％,較正常對照組(100.00％±5.28％)明顯增加(P =0.000),H2O2+Tβ4組細胞內ROS水平(163.26％±10.53％)雖然較正常對照組偏高,但與H2O2組(234.42％ ±22.15％)比較顯著降低(P=0.000).TUNEL染色結果顯示,H2O2組呈現較多凋亡細胞,H2 O2+ Tβ4組與H2O2組相比,凋亡細胞明顯減少.結論 Tβ4通過抑製氧化損傷後細胞內ROS的產生對氧化應激損傷後的角膜上皮細胞具有減少細胞凋亡,增加細胞存活率併促進細胞遷移的作用,提示Tβ4對角膜上皮細胞氧化損傷具有保護作用.
목적 탐토흉선소β4(Tβ4)대체외배양적토각막상피세포양화손상적영향.방법 실험연구.채용조직괴배양법획득원대토각막상피세포병진행전대배양.채용형태학관찰화역전록취합매련반응(RT-PCR)법검측성숙각단백(keratin) 12화봉극련접단백(connexin) 43적표체정황,대세포진행감정.이H2O2처리각막상피세포제비체외세포양화손상모형,병관측Tβ4대기보호작용.실험분위4조:정상대조조、Tβ4조、H2O2조화H2O2+ Tβ4조.채용MTT비색법검측각막상피세포존활도；운용TUNEL염색관찰각막상피세포조망；이용DCFH-DA형광탐침검측세포내활성양(ROS)수평；통과화흔실험관찰각막상피세포적천이운동능력.조간균수비교채용방차분석,진일보량량비교채용LSD-t검험.결과 배양적세포정원형、란원형화다변형,포로석양생장；RT-PCR법검측현시배양적세포표체각막상피세포특이성기인,keratin 12화connexin 43,정현각막상피세포적특정.경H2O2자격후,각막상피세포적존활솔(67.20％±5.87％)교정상대조조현저하강(l00.00％±9.99％)(4개조간비교F=18.61,P=0.001,량량비교P=0.000)；H2O2+Tβ4조세포존활솔(83.42％±7.43％)여H2O2조(67.20％±5.87％)비교명현증고(P =0.023).4개조간세포화흔실험결과적차이구유통계학의의(F =36.38,P=0.000),재12 hTβ4조화흔이기본소실,세포천이솔위117.6％ ±2.22％,여정상대조조(100.00％±4.06％)상비현저증가(P=0.005)；12h시H2O2+Tβ4조여H2O2조상비,세포천이솔위96.57％±8.22％,여H2O2조(64.38％±11.08％)상비명현증가(P=0.000).H2 O2자격후세포내ROS수평위234.42％±22.15％,교정상대조조(100.00％±5.28％)명현증가(P =0.000),H2O2+Tβ4조세포내ROS수평(163.26％±10.53％)수연교정상대조조편고,단여H2O2조(234.42％ ±22.15％)비교현저강저(P=0.000).TUNEL염색결과현시,H2O2조정현교다조망세포,H2 O2+ Tβ4조여H2O2조상비,조망세포명현감소.결론 Tβ4통과억제양화손상후세포내ROS적산생대양화응격손상후적각막상피세포구유감소세포조망,증가세포존활솔병촉진세포천이적작용,제시Tβ4대각막상피세포양화손상구유보호작용.
Objective The aim of this study was to investigate the effects of thymosin β4 (Tβ4)against oxidative damage in rabbit corneal epithelial cells in vitro.Methods Experimental study.Primary cultures of rabbit corneal epithelial cells were isolated and cultured from cornea tissue explants.Cell morphology was observed by phase-contrast microscope.The expression of keratin 12 and connexin 43 in cultured cells were examined with RT-PCR.The cultures were divided into 4 groups:control group,Tβ4 group,hydrogen peroxide (H2O2) group,and H2O2 + Tβ4 group.The cell viability of cultured corneal epithelial cells was examined by MTT assay.TUNEL staining was used to detect the apoptotic cells.ROS levels were estimated by DCFH-DA using fluorescent microscopy.Cell migration was measured using the scratch wound technique.Data were analyzed using one-way analysis of variance (ANOVA) and secondary analysis for significance with post Hoc tests.Results The morphology of cultured cells was round,ovoid,polygonal or paving stone appearance.The results of RT-PCR showed that cultured corneal epithelial cells expressed both keratin 12 and connexin 43,the characteristic genes of corneal epithelial cells.The cell viability in H2O2 group was significantly reduced than that in control group (67.20％ ± 5.87％ vs.100.00％ ±9.99％,P=0.000); the cell viability was significantly improved in Tβ4 + H2O2 group than that in H2O2 group (83.42％ ± 7.23％ vs.67.20％ ± 5.87％,P =0.023).Cell migration was significantly increased in Tβ4 group compared with the controls (117.6％ ± 2.22％ vs.100.00％ ±4.06％,P=0.005); Compared with H2O2 group,cell migration was significant increase in H2O2 + Tβ4 group (96.57％ ±8.22％ vs.64.38％ ± 11.08％,P =0.000).Compared with the control group,the intracellular ROS level of the H2O2 group was significantly increased (234.42％ ±22.15％ vs.100.00％ ± 5.28％,P =0.000).Intracellular ROS level of H2O2 + Tβ4 group was significantly decreased as compared with the H2O2 group (163.26％ ± 10.53％ vs.234.42％ ±22.15％,P=0.000).TUNEL staining indicated that Tβ4 treatment markedly inhibited H2O2-induced apoptosis in cultured corneal epithelial cells.Conclusions Tβ4 has a strong protection effect against oxidative damage induced by H2O2 in corneal epithelial cells through promoting cell growth and cell migration,and the underlying mechanisms may due to the antioxidation and anti-apoptotic effects of Tβ4.
