ABSTRACT
Background Laser assisted subepithelial keratomileusis (LASEK) is one of surgical procedures for refractive correction.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 Tβ4 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 cells were regularly cultured in the normal control group, and Tβ4 was added in the culture medium at the final concentration of 1 μg/ml 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 μg/ml in the model+Tβ4 group.The survival rate of the cells was detected by MTT assay, and the apoptosis rate of the cells 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 flurescenee quantitative PCR.The content of bcl-2 protein in the cells was detected by ELISA assay.Spectrophotometry 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+Tβ4 group (P=0.001 ,P=0.035).TUNEL staining revealed more positive cells in the model group and the model+Tβ4 group,and the percentage of TUNEL positive cells was (30.0±6.7)％ in the model+Tβ4 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 ethanoldamaged rabbit corneal epithelial cells.In addition, Tβ4 promotes the regrowth of corneal epithelial cells by upregulating the expression of cyclin D1 and CDK4.
ABSTRACT
Background Oxidative stress is a pathophysiological process of retina,so it is very important to explore a protective way against retinal oxidative stress.Studies determined that extract of ginkgo biloba (EGb) has antioxidant,anti-apoptosis,anti-thrombosis and anti-inflammatory effects,however,the effect of EGb on human Müller cells in oxidative stress is still below understood.Objective This study was to investigate the protection of EGb against oxidative stress of human retinal Müller cells induced by As2O3 in vitro.Methods Human retinal Müllercell line was cultured in DMEM/F12 with 10％ fetal bovine serum (FBS),2 μmol/L glutamine and 1％ antibiotics.As2O3 solution at the final concentration 5 mg/L was added in the medium for 24 hours to establish oxidative models,and then the EGb with the final concentrations of 5,10 and 20 mg/L was used to cell models for 24 hours,respectively.Cell viability was detected by MTT assay,and reactive oxygen species (ROS) levels in the cells were detected with CM-H2DCFDA fluorescent probe.The relative expression levels of caspase-3 mRNA in cytoplasm and cell nuclei were assayed by reverse-transcription PCR (RT-PCR),and the expressions of Nrf2 protein were quantitatively detected by Western blot.Results Müller cells adhered well 24 hours after cultured.At 6-7 days after culture,Müller cell body was large with abundant cytoplasm and mosaic-like arrangement.However,floating cells were seen after As2 O3 treatment.Cell viability (absorbance) was significantly different among the normal culture group,As2 O3-treated group,As2 O3 + 5 mg/L EGb group,As2 O3 + 10 mg/L EGb group and As2 O3 + 20 mg/L EGb group,with the strongest viability in the normal culture group and the weakest viability in the As2 O3-treated groups (F=163.57,P =0.00).The fluorescence intensity of ROS was the weakest in the normal culture group and the strongest in the As2 O3-treated group and was gradually weakened with the increase of EGb doses,showing a remarkable difference among the groups (F =4 013.61,P =0.00).The relative expression level of caspase-3 mRNA in the cells was gradually reduced with the increase of EGb doses,with a statistically significant difference among the groups (F =2 199.72,P =0.00).In addition,no considerable difference was seen in the expression level of Nrf2 protein (grey scale) in cytoplasm among the groups (F=15.42,P=0.40);while in the nuclei,the expression levels of Nrf2 protein were 100.01 ±0.04,46.59±0.63,54.51 ±0.62,59.93 ±0.17 and 67.60±0.24 in the normal culture group,As2 O3-treated group and As2O3+5 mg/L EGb group,As2O3+10 mg/L EGb group,As2O3+20 mg/L EGb group respectively,with a significant difference among them (F=7 271.72,P=0.00).Conclusions EGb can protect human retinal Müller cells against As2O3-induced damage in a dose-dependant manner by antioxidant and antiapoptotic effects in vitro,and the activities occur primarily in cell nucleus.
ABSTRACT
Background Limbal stem cells (LSCs) play an important role on the stability of corneal epithelium and corneal transparency.Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor can promote cell proliferation and reduce apoptosis,such as human embryonic stem cells and karatin epithelial cells.Objective This study was to investigate the improving effect of Y-27632,a ROCK inhibitor,on the activity of rabbit LSCs in corneal preservation medium.Methods Corneal preservation solution was prepared by adding 12.5％ chondroitin sulfate,10.0％ low molecular dextran,20.0 mg/L dexamethasone,100 mg/L tobramycin sulfate,9.5 g/L Hepes and 0.375 mg/L L-glutamine in MEM.The corneas of New Zealand white rabbits were collected and preserved in the corneal preservation solution with or without Y-27632 for 4,7,14 days,and the density and morphology of corneal endothelial cells were examined by using 0.25％ trypan blue staining and 0.2％ alizarin red staining.Isolated corneal epithelial cells were seeded on 3T3 feeder layer and cultured for 7-10 days until colonies formation.Colony shape of LSCs was observed under the light microscope,and colony-formation efficiency was analyzed after Giemsa staining by Image J software.Results The morphology and density of corneal endothelial cells were normal in the corneal preservation solution with and without Y-27632 for 4 days.In the seventh day after preservation,the cells remained the regular hexagon in shape in the preservation solution with Y-27632,however,the cellular membrane was slightly shrinking with the positive staining for alizarin red in the preservation solution without Y-27632.The density of corneal endothelial cells in the corneal preservation solution without Y-27632 was (2 262-± 75) cells /mm2,while in the preservation solution with Y-27632 was (2 425 ±95) cells/mm2(P＜0.001).The cloning spheres of LSCs were similar in preservation solution both with and without Y-27632 in the freshly isolated cornea or preserved corneas and exhibited more cells inside.But in 7 days and 14 days after preservation,the cloning spheres were much smaller in the preservation solution without Y-27632 group than those in the preservation with Y-27632 group.No significant differences were found in the cloning-formation rate and survival rate of corneal epithelial cells in corneas freshly isolated or preserved for 4 days in both groups (all at P＞0.05).In 7 days and 14 days after preservation,the active rates of corneal epitheli.al cells were (73.00±2.12)％ and (56.00±0.71)％ in the preservation solution with Y27632,which were significantly higher than (66.00 ± 4.00) ％ and (49.00 ± 0.71) ％ in the preservation solution without Y-27632,showing statistically significant differences between them (t =3.098,P =0.018;t =9.798,P =0.000).In addition,the cloning-formation rates of LSCs were (11.05±0.21)％ and (3.10±1.97)％ in the preservation solution with Y-27632 in 7 days and 14 days after preservation,revealing significantly elevation in comparison with (2.05 ± 1.20) ％ and (0.40 ±0.14) ％ in the preservation solution without Y-27632 (t =18.107,P =0.000;t=3.184,P=0.017).Conclusions Y-27632 promotes the vitality and cloning-formation ability of LSCs in corneal preservation medium,suggesting its potential use during storage of cornea.