胸腺素β4对体外培养的兔角膜上皮细胞氧化损伤的保护作用

摘要

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.%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.