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  

Protective effect of thymosin β4 against oxidative damage in cultured rabbit corneal epithelial cells

作　　者：李轩[1] 姜志昕[1] 郝朋[1] 汤欣[1] 

机构地区：[1]天津市眼科医院、天津市眼科学与视觉科学重点实验室、天津市眼科研究所、天津医科大学眼科临床学院

出　　处：《中华眼科杂志》2013年第8期716-722,共7页Chinese Journal of Ophthalmology

基　　金：国家自然科学基金项目（81170828）;天津市应用基础与前沿技术研究计划项目（11JCYBJC27200）

摘　　要：目的探讨胸腺素B4（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H_20_2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、H20：组和H_2O_2＋Tβ4组。采用MTF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，keratin12和connexin43，呈现角膜上皮细胞的特征。经H_2O_2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（100．00％±9．99％）（4个组间比较F=18．61，P=0．001，两两比较P=0．000）；H202＋Tβ4组细胞存活率（83．42％±7．43％）与H202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12hTβ4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H_20_2＋Tβ4组与H202组相比，细胞迁移率为96．57％±8．22％，与H202组（64．38％±11．08％）相比明显增加（P=0．000）。H202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），H_20_2＋Tβ4组细胞内ROS水平（163．26％±10．53％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 （ H： 02 ） group, and H2 02 ± Tβ4 group. The cell viability of cultured cornealepithelial cells was examined by MTY 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 resuhs 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 H202 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 ± H202 group than that in H202 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 H202 group, cell migration was significant increase in H202 ± 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 H202 group was significantly increased （234.42% ±22. 15% vs. 100. 00% ± 5.28% , P = 0. 000）. Intracellular ROS level of H202 ± Tβ4 group was significantly decreas

关 键 词：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 

分 类 号：R772.2[医药卫生—眼科]