胸腺素β4对乙醇诱导的体外兔角膜上皮细胞损伤的保护作用

Protective effects of thymosin Tβ4 on ethanol-induced rabbit corneal epithelial cell damage in vitro

背景准分子激光角膜上皮瓣下磨镶术（LASEK）是目前主要的屈光矫正术式之一，但术中使用的乙醇可引起角膜上皮的损伤。胸腺素β4（Tβ4）具有抗凋亡等多种生物学作用，但其对乙醇所致的角膜上皮细胞损伤是否具有保护作用尚不清楚。目的研究T[34对乙醇诱导的角膜上皮细胞损伤是否具有预防作用。方法收集10只健康成年新西兰白兔的角膜，采用组织块培养法获得兔原代角膜上皮细胞传代，逆转录PCR（RT—PCR）法检测缝隙连接蛋白COilnexin 43和成熟角蛋白keratin 12的表达情况，对细胞进行鉴定。选取第2代处于对数生长期的细胞均衡分为4个组，正常对照组细胞进行常规培养，Tβ4组细胞在培养液中添加终浓度为1μg／ml的Tβ4，乙醇组细胞用含体积分数20％乙醇的PBS作用20s以制备角膜上皮细胞损伤模型，乙醇＋Tβ4组在损伤模型培养液中加入Tβ4。采用MTT法检测各组角膜上皮细胞存活率；采用TUNEL法观察角膜上皮细胞的凋亡情况；采用实时荧光定量PCR法检测各组角膜上皮细胞中细胞周期蛋白D1（CyclinD1）和细胞周期蛋白依赖性激酶4（CDK4）mRNA的相对表达量；应用ELISA法检测各组角膜上皮细胞中bcl-2蛋白的质量浓度；用分光光度法检测细胞裂解液中caspase-3酶活性。结果MTT法结果显示，正常对照组细胞存活率设定为100％，乙醇组细胞存活率为（52．1±14．07）％，明显低于正常对照组，差异有统计学意义（P=0．001）；乙醇＋TB4组细胞存活率为（77．7±19．60）％，明显高于乙醇组，差异有统计学意义（P=0．035）。TUNEL染色显示，乙醇组和乙醇＋Tβ4组均可见明显的TUNEL阳性染色细胞，乙醇＋Tβ4组阳性细胞百分比为（30．0±6．7）％，明显低于乙醇组的（42．4±4．0）％，差异有统计学意义（P=0．01）。实时定量PCR显示，乙醇＋Tβ4组细胞中Cyclin D1 mRNA和CDK4 mRNA的相对表达量分别为0．93±0．17和0．88±0．09，均明显高于乙醇组的0．68±0．05和0．54±0．07，差异均有统计学意义（P=0．027、0．002）。ELISA显示，乙醇组细胞中bcl-2蛋白质量浓度明显低于乙醇＋Tβ4组，而caspase-3酶活性则高于乙醇＋Tβ4组，差异均有统计学意义（P=0．030、0．021）。结论Tβ4对乙醇诱导的角膜上皮细胞损伤具有保护作用，其作用机制可能是通过降低角膜上皮细胞中caspase-3酶活性和增加bcl-2蛋白质量浓度来阻止乙醇所致的细胞凋亡；Tβ4也可上调角膜上皮细胞中Cyclin D1及CDK4的表达，以调控细胞分裂和增生，促进角膜上皮损伤的愈合。

Background Laser assisted subepithelial keratomileusis （LASEK） is one of surgical procedures for refractive eorreetion. Dilute alcohol that is used for the removal of epithelium during LASEK induces the apoptosis of corneal epithelial cells. Researches showed that thymosin β4 （Tβ4） can arrest apoptosis, but whether Tβ4 plays inhibitory effect on ethanol-induced damage of corneal epithelial cells is still unelucidated. Objective The aim of this study was to investigate the protective effects of TI34 on ethanol-induced rabbit corneal epithelial cell damage in vitro. Methods The corneal tissue of deendothelium was obtained from 10 New Zealand white rabbits. Corneal epithelial cells were cultured in vitro by using explant culture method. Cultured cells were identified by detecting the expression of keratin 12 and connexin 43 with reverse transcription PCR （RT-PCR）. The cells of second generation were collected and divided into 4 groups. The ceils were regularly cultured in the normal control group, and T134 was added in the culture medium at the final concentration of 1 ixg/m] in the Tβ4 treated group. Ethanol-induced rabbit corneal epithelial cell damage models were established by adding PBS containing 20% alcohol in medium for 20 seconds in the model group,and Tβ4 was added in the medium of model cells at the final concentration of 1 ixg/ml in the model＋Tβ4 group. The survival rate of the cells was detected by MTT assay, and the apoptosis rate of the ceils was examined by TUNEL method. The relative expression levels of cyclin D1 mRNA and cyclin-depensent protein kanase 4 （CDK4） mRNA in the cells were detected by real-time flurescence quantitative PCR. The content of bcl-2 protein in the ceils was detected by ELISA assay. Speetrophotometry was employed to measure the activity of caspase-3. The study complied with the Regulation for the Adminstration of Affairs Concerning Experimental Animals by State Science and Technology Commission. Results The mean cell survival rate was （52. 1 ± 14. 07 ） % in the model group, which was significantly reduced in comparison with 100% of the normal control group and （77.7± 19.60） % of the model＋TI34 group （P = 0.001 ,P =0.035 ）. TUNEL staining revealed more positive ceils in the model group and the model＋TI34 group,and the percentage of TUNEL positive cells was （30.0±6.7）% in the model＋T[34 group,showing an evident decline in comparison with （42, 4±4.0）% of the model group （P=0.01）. The relative expression levels of cyclin D1 mRNA and CDK4 mRNA were 0.93±0. 17 and 0.88±0.09 in the model＋Tβ4 group, which were significantly higher than 0.68±0.05 and 0. 54±0.07 in the model group （P = 0. 027,0. 002）. ELISA assay exhibited that bcl-2 content in the cells was considerably lower, and caspase-3 activity was significantly higher in the model group than those in the model＋Tβ4 group （ P = 0. 030,0. 021 ）. Conclusions Tβ4 plays a protective effect on rabbit corneal epithelial cells from apoptosis by lowing the caspase 3 activity and increasing bcl-2 content in ethanol- damaged rabbit corneal epithelial cells. In addition, Tβ4 promotes the regrowth：of corneal epithelial cells by up- regulating the expression of cyclin D1 and CDK4.