| ObjectivesTo explore low level laser irradiation(LLLI)bone marrow mesenchymal stem cells(marrow mesenchymal stem cells),BMSCs)contribute to bone differentiation by activating AMPK-SIRT1 signaling pathway.Methods1.BMSCs were cultured in rats and identified by flow cytometry.2.According to the results of the preliminary experiment,the energy density was set as 2 J/cm~2,and the LLLI with different power densities of 0,0.04,0.08,0.16,0.32,0.5 and 1.0 W/cm~2were used to irradiate rat BMSCs.The proliferation capacity of BMSCs was detected by CCK-8 experimenter-1,and the optimal power density was screened.3.The results showed that the optimal power density was 0.04 W/cm~2,and the energy densities were set as 0,2,4,8,10,12,14,16 J/cm~2LLLI to irradiate BMSCs.The proliferation capacity of BMSCs was detected by CCK-8 experimenter 2,and the optimal energy density was screened.4.BMSCs of rats were irradiated with LLLI of 0.04 W/cm~2and energy densities of 0,2,4 and 8 J/cm~2,respectively.Alizarin red staining,ALP activity detection,RT-qPCR and immunofluorescence were performed to detect the expression of genes and proteins related to osteogenic differentiation.5.With a power density of 0.04 W/cm~2and an energy density of 0 and 8 J/cm~2,LLLI irradiated rat BMSCs,underwent alizarin red staining,and conducted ALP activity test.m RNA and protein expression levels of osteogenic factors,AMPK,p-AMPK and SIRT1 were detected by immunofluorescence,RT-qPCR and WB.Results1.Flow cytometry showed that CD44,CD29 and CD90 were expressed positively,while CD45 was expressed negatively,and the purity of BMSCs was90.73%.2.The results of CCK-8 showed that when the power density was 0.04 W/cm~2and the energy density was 8 J/cm~2,the cell proliferation activity was the highest.3.Compared with the control group,the content of calcium nodules and the activity of ALP were increased in the laser group,and the m RNA and protein expressions of COL-I,OPN,RUNX2,OCN and SIRT1 were enhanced in the laser group,and the phosphorylation of AMPK was elevated,with statistical difference(P<0.05).ConclusionsThe 808 nm near infrared laser promoted the proliferation and osteogenic differentiation of BMSCs by activating AMPK-SIRT1 signaling pathway,and the effect was most significant when the power density was 0.04W/cm~2and the energy density was 8 J/cm~2. |
