Low Level Semiconductor Laser Regulates Human Tenon's Capsule Low Level Semiconductor Laser Regulates Human Tenon's Capsule Fibroblasts Proliferation And Migration Introduced By Thymosin?4

ObjectiveTo study the effects of low-level laser therapy(LLLT)on proliferation and migration of human tenon's capsule fibroblasts(HTFs).To determine the optimal laser irradiation parameters of LLLT influencing the proliferation and migration of human tenon's capsule fibroblasts.To investigate the regulation of thymosin?4(T?4)expression in human tenon's capsule fibroblasts by low energy laser therapy and the mechanism of its effect on proliferation and migration of human tenon's capsule fibroblasts.Methods1.Establish LLLT-induced HTFs cell line modelHTFs were obtained from patients with cataract during surgery.human tenon capsule tissue was cultured into fibroblasts,and primary HTFs were cultured in vitro.after passage,5~10 passages of HTFs were used for experiment.They were induced by different level laser for different durations.Different laser dose groups(0,0.5,1.0,2.0,5.0J/cm~2)and non-irradiated group(control group)were used to select the optimal irradiation dose of LLLT to promote HTFsgrowth and viability by CCK8 method.The optimal irradiation parameters of LLLT on HTFs were selected by setting the same laser dose group and using circular homogeneous light spots with diameters of 10 mm and 35 mm respectively.2.Detection the effects of LLLT on proliferation and migration of HTFsCCK8 and cell migration assays were performed with fibroblasts after LLLT with a semiconductor laser at various doses(0,0.5,1.0,2.0,or 5.0 J/cm~2)to observe the effects of LLLT with a semiconductor laser on the proliferation and migration of fibroblasts.The non-irradiated group served as the control.3.Detect the expression level of T?4 in LLLT-induced HTFsMoreover,western blot analysis and immunofluorescence were performed with fibroblasts after LLLT with a semiconductor laser to analyze changes in the activities of thymosin?4.While the gene expression was detected by real-time quantitative PCR.Results1.In vitro cultured HTFs cells successfully.The cultured cells were active growing,stable in manifestation,and confirmed to be fibroblast.2.different doses of LLLT had dose-dependent effect on cell growth,that is,HTFs growth accelerated in a certain range of laser irradiation dose,and HTFs growth was the fastest under LLLT irradiation of 2.0J/cm~2,the difference was statistically significant compared with the control group(p<0.01).3.At the same dose of LLLT,the growth activity of HTFs was different with different light spot size.A circular uniform spot with a diameter of 10 mm is better than a 35 mm spot.4.the model of HTFs induced by LLLT was successfully established:the power density of low energy semiconductor laser irradiation system was set to 20 mw/cm~2.Power densities were generated in a round homogeneous spot with a diameter of10 mm.The low level semiconductor laser system was used with continuous wave mode and following irradiation power(irradiance,irradiation time);0.5 J/cm~2(20mw/cm~2,25 sec),1.0 J/cm~2(20 mw/cm~2,50 sec),2.0 J/cm~2(20 mw/cm~2,20 sec),5.0J/cm~2(20 mw/cm~2,250 sec).5.In CCK8 method,transwell assays and scratch experiment,it was shown that the proliferation and migration of HTFs were promoted by 2.0J/cm~2 low-level semiconductor laser.6.Western blot analysis revealed that LLLT with semiconductor laser had a bi-directional effect on the expression of T?4,and the optimal dose was 2.0J/cm~2,which was significantly different from the control group(0J/cm~2)(p<0.05).In addition,a single product was obtained by RT-PCR amplification.Conclusion1.Low energy semiconductor laser promote that proliferation and migration of human tenon's capsule fibroblasts;2.Activation of T?4 may be a necessary condition for low energy semiconductor laser induced proliferation and migration of fibroblasts;3.Low energy semiconductor laser may regulate T?4 to promote proliferation and migration of human tenon capsule fibroblasts.