Abstract:Objective To investigate the effect of histone deacetylase 2 (HDAC2) on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts based on the Hedgehog signaling pathway. Methods BMSCs were isolated and cultured in vitro and identified by morphological observation and flow cytometry. Transfer to the third generation of BMSCs cells, and use liposome transfection to transfer the HDAC2-siRNA recombination sequence and negative random control sequence into the cells respectively, and name them HDAC2-si group and no-load group, and blank cells without intervention group The expression of HDAC2 mRNA in blank group, empty group and HDAC2-si group 48 hours after transfection was detected by real-time fluorescence quantitative PCR. Dexamethasone induced the cells to differentiate into osteoblasts. After 7 days, Western blot was used to detect the expression of HDAC2 protein. The ALP activity was detected by p-nitrobenzene method, and the amount of osteocalcin (BGP) was measured by isotope labeling. Alizarin red staining to observe the effect of transfection on osteogenic differentiation of BMSCs. Western blot was used to detect the protein expression of SHH, IHH, Ptc1 and Glis1 in Hedgehog signaling pathway. Results BMSCs cells grew in polygonal or spindle shape in primary culture for one day, and in colony shape after three days. Then they began to proliferate rapidly. At the sixth day, the cells fused to more than 90% and grew in spiral or whirlpool shape. There was no significant change in cell morphology from subculture to the third generation. The surface markers of BMSCs in the third generation were identified by flow cytometry to be in line with the biological characteristics of BMSCs. Forty-eight hours after BMSCs transfection, the relative expression of HDAC2 mRNA in HDAC2-si group was lower than that in blank group and empty group (P < 0.05), while there was no significant difference between blank group and empty group (P > 0.05). The results of alizarin red staining showed that the three groups before induction were spiral or vortex without red mineralized nodules, after 7 days of induction, red mineralized nodules appeared in the blank group and the no-load group, and the HDAC2-si group had deeper staining and more red mineralized nodules. Compared with the blank group and the empty group, the relative expression of HDAC2 protein was lower after 7 days of bone induction, ALP activity after 7 days, BGP secretion after 14 days and SHH, IHH, PTCH1, Glis1 protein were higher in HDAC2-si group (P < 0.05), while there was no significant difference in the relative expression of IHH protein between groups (P > 0.05). There was no significant difference in ALP activity, BGP secretion and relative expression of HDAC2, SHH, IHH, PTCH1 and glis1 between the blank group and the empty group (P > 0.05). Conclusion Down-regulating the expression of HDAC2 can promote the differentiation of BMSCs into osteoblasts, which may play a role in promoting osteogenic differentiation by activating Hedgehog signaling pathway related molecules.