| BackgroundLow-level laser(low-level laser,LLL)is a series of laser,the wavelengths at 600-1100nm,energy density 1-10J/cm2.power density of 3-90mW/cm2.Low-level laser treatment(LLLT)can not only promote tissue repair and regeneration,but also enhance the activity of cultured cells in vitro.Studies have shown that LLLT played a key role in cell proliferation and migration in vitro.However,the current study has not yet clearly illustrated the optimal parameters for cell proliferation.In addition,since LLLT has proved to have the effect on promoting hair growth,it has been used for lopecia treatment.However,the mechanism of these biological effects remains unclear,which becomes a factor limiting the application of LLLT.Our study aims to explore the optimal parameters of LLLT in promoting cell proliferation in vitro and to explore its underlying mechanism for promoting hair regrowth,laying a theoretical foundation for a broader clinical application of LLLT.Objectives1.To study the effects of LLLT on the culture of mesenchymal stem cells(ADSCs)and Bone marrow derived stem cells(BMSCs),and its optimal parameters for cell proliferation of cultured MSC cells.2.To explore the effects of LLLT on hair follicles in the skin of C3H/HeJ mice and its underlying mechanism.Methods and Results1.Detection of proliferation,cell cycle and apoptosis of stem cells(SCs)in vitro.Methods:Human ADSC cells and rat BMSC cells were harvested and were cultured in mesenchymal stem cell culture medium.After the cells were well adhered,the low,medium and high dose LLLT irradiation was given to the two types of cells respectively.The irradiation time were 39s,196s and 392s respectively.The cell viability was measured by MTS method at 6h,12h,24h and 48h after irradiation.The cell cycle and apoptosis were detected by FACS technique.We evaluated the effects of LLLT on the biological characteristics of human ADSCs and rat BMSCs in vitro and compared the different effects caused by different dose of irradiation,furthermore,defined the optimal parameter of LLLT for SCs cell proliferation.Results:(1).LLLT has an energy-dependent effects on the proliferation of SCs cultured in vitro.LLLT can promote the proliferation of SCs within a certain range,however,this promoting effect was not significant when the dose of LLLT was beyond this energy range.(2).The effect of LLLT on the proliferation of SCs in vitro was different for different cells.For rat BMSCs and human ADCSs.the difference was observed in the onset of cell proliferation.(3).High dose of LLLT can increase the level of apoptosis in SCs cultured in vitro(rat BMSCs,human ADSCs).2.Detection of migration ability of human ADSCs and rat BMSCs in vitro.Methods:Human ADSCs and rat BMSCs were harvested and the cells were seeded in a six-well plate.After the cells growth of 80%-90%fusion,the cells were used for experiment.Scratches were made in the six-well plate.The wells were cultured in a 37°C,5%CO2 incubator.6h,12h,24h,48h after sampling,the statistic analysis was performedResults:(1).Low dose of LLLT could enhance the migration ability of human ADSCs cells,however,the medium and high dose of LLLT did not weaken the migration of ADSCs.(2).Low dose of LLLT had no effect on the migration ability of rat BMSCs.The medium and high dose of LLLT attenuated the migration ability of rat BMSCs.3.Effects of LLLT on hair follicles in C3H/HeJ mice and the underlying mechanism.Methods:Female C3H/HeJ mice were randomly divided into two groups:laser irradiation group(LLLT group),control group(control group),all mice were depilated gently.Intraperitoneal injection of EdU solution was performed followed by LLLT irradiation.The laser device was gently appled to the mouse skin.Mice were sacrificed immediately after 4 h,1 d,2 d,5 d,7 d,10 d,15 d,20 d after LLLT irradiation.The results of H&E staining,immunohistochemistry staining,rt-PCR and western blot results were statistically analyzed.Results:LLLT had the effect on inducing the telegon hair follicles in the dorsal skin of C3H/HeJ mice to re-enter the anagen phase.This effect was achieved by activating the Wnt 10b-mediated Wit10b/β-catenin signaling pathway.Conclusions1.Medium and low energy LLLT has the effect on inducing GO/G1 phase cells into the S phase,and few effect on promoting proliferation and induction of apoptosis.High dose of LLLT can not increase the proliferation rate of SCs,whereas,it increases the level of cell apoptosis.(2).As for the ability of cell migration,for BMSCs,when the dose is high,LLLT limits the migration of BMSCs,and low doses has no significant effect.For ADSCs,low-dose of LLLT enhances cell migration,whereas,medium and high doses of LLLT do not diminish the migration ability of ADSCs.(3).LLLT can promote the regrowth of hair follicles in mice,since it can induce the telegon-anagen-transition,in the meanwhile,it promotes the proliferation of hair follicle cells.The promoting effects were achieved by up-regulating the key proteins in Wnt10b/β-catenin signaling pathway. |
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