The Effect Of Thymosin Beta4on Proliferation,Migration And Epithelial-mesenchymal Transition Of Human Lens Epithelial Cells In Vitro

Purpose:Posterior capsule opaciftcation (PCO) is caused mainly by the proliferation, migration and epithelial-mesenchymal transition (EMT)of human lens epithelial (HLE) cells. Thymosin beta4(Tβ4) Tβ4is an important regulator in actin polymerization and repolymerization which is essential for cytoskeleton organization according to cellular needs. This study investigated the role of Tβ4in PCO formation by observing effect of thymosin beta4on proliferation, migration and epithelial-mesenchymal transition of human lens epithelial cells in vitro.Methods1. The effect of Tβ4on proliferation of HLEC SRA01/04was detected by MTT assay. The HLEC SRA01/04were collected and used in this experiment at the concentration of1×104cells/hole. The serum-free DMEM containing lng/ml10ng/ml、100ng/ml、1000ng/ml of Tβ4was added into medium for24hours,48hours and72hours in different experimental group respectively, and only equal volume of free-serum DMEM was added in control group.2. HLEC SRA01/04migration ability was evaluated by transwell migration and wound healing assays.(1)The LECs were treated with Tβ4by different concentrations(0, lng/ml、10ng/ml、100ng/ml、1000ng/ml) in vitro wound healing model. Serial images of10times in random fields of microscope were captured immediately at0、12h and24h after scratching using an image analyzer. Scratch width was measured respectively. The migration distance at different time points were calculated according to the following formula:Width of the initial scratch—Width of the scratch at corresponding point.(2)In transwell migration model,1×105cells/well were seeded into the upper polycarbonate membrane insert (pore size,8μm) whereas800μl of cultured medium with20%fetal bovine serum containing Tβ4at lng/ml、10ng/ml、100ng/ml、1000ng/ml was added to the lower chamber, wells containing serum-free medium without Tβ4served as controls. After24h of incubation,the membranes were fixed and stained, then scraped off the cells in upper insert with a cotton swab. The number of migratory cells was counted five times in random fields of microscope.3. To examine the role of Tβ4in regulating LECs epithelial-mesenchymal transition (EMT), we tested the mRNA of a-smooth muscle actin (aSMA) and E-cadherin by real-time PCR assay, with and without Tβ exposure for72h.Results1. HLECs proliferation was markedly increased in100ng/ml and1000ng/ml Tβ4treated groups in a time-dependent manner, as compared with that in control group(P<0.05)2. The average migration distance of wound healing assay at24h in different concentrations were329.6621±47.43715μm,354.2761±52.5730μm,453.5897±46.3792μm,499.6127±46.0270μm,555.4763±71.6129μm; while that at48h were614.6647±97.2274μm,636.1453±649.7268μm,720.5249±41.9510μm,739.9166±85.3627μm,770.9152±62.3144μm. The migration distance of LECs in10ng/ml、100ng/ml、1000ng/ml Tβ4was significantly increased compared with that of control cells (p<0.05).Moreover, HLE migration ability increased in a dose-and time-dependent manner. With Tβ4treatment varying from concentrations of Ong/ml to1000ng/ml, the amount of cells transwell were82±3,109±6,128±10,163±5,204±11, respectively. The effect of Tβ4on LECs migration was enhanced with the increase of Tβ4concentration (P <0.05).3. Compared with control group, the expression of lens epithelial marker E-cadherin mRNA in experimental group was significantly down-regulated in LECs cells(P<0.05), whereas that of the mesenchymal marker a-SMA was upregulated(P<0.05).ConclusionThymosin beta4can induce proliferation, migration, and EMT of HLE cells and may be a valuable therapeutic target for the prevention and treatment of PCO.