Effects Of TLR2 Ligand Peptidoglycan On Human Mesenchymal Stem Cells Proliferation And Differentiation Into Adipocytes And Osteoblasts

Objective The study was aim to explore the effect of peptidoglycan (PGN) on the proliferation and differentiation of human bone marrow mesenchymal stem cells (BM-MSC) into Adipocytes and Osteoblasts.Methods BM-MSC was isolated from bone marrow of healthy donor by Ficoll method. Expression of CD34, CD45, HLA-DR, CD44 and CD71 in BM-MSC were shown by flow cytometry. The proliferation of cells was tested with MTT assay, and then the cell growth curve was drawn according to optical density. Cell cycle was measured by flow cytometry assays. In the Adipocytes and Osteoblasts differentiation induction medium, their effects on MSC were observed.Results The cells shape was observed by inverted microscope, We found that the BM-MSC were homogeneous,spindle-shaped and fibroblast-like cells. The cells can be propagated and extensively amplified in vitro. The Results showed that high expression of CD44 and CD71 in BM-MSC, while expression of CD34, CD45 and HLA-DRwas negative by FCM. The results showed that the cell proliferation index was significantly increased in a dose and time dependent manner after BM-MSCs was incubated with PGN, (P<0.05). The effects on the proliferation of BM-MSCs were highest in 10ug/mL group, 10ug/m,20ug/ml was no significant difference(P>0.05). Compared with the control group, PGN could significantly decrease G0/G1 ratio and increase S and G2/M stage ratio(P<0.05). In the Adipocytes differentiation induction medium, After being induced with 72 hours, the cells containing lipid droplets appear to extend over time, lipid droplets, and the gradual integration of the lipid vesicle aggregation. the Adipogenic rate of BM-MSC were 65.5%±5.3%,85.2%±3.4% in 14,21 days. While the experimental group were 32.2%±4.8%,42.3%±3.4%,(P<0.05). In the Osteoblasts differentiation induction medium, ALP was began to express on day 7 and Mineralized nodules in the 10th day of induction, the Alizarin red of BM-MSC were 70.5%±3.5%,87.5%±3.5% in 14,21 days. While the experimental group were42.5%±6.5%,60.5%±3.5%, (P<0.05), (P<0.05). Conclusion PGN can promote more BM-MSCs to enter the stage of DNA synthesis and produce many proliferative cells.In addition, this effect is in a dose and time dependent manner. Peptidoglycan inhibit BM-MSC differentiated into Adipogenic and Osteoblasts, TLR-2 may be involved in regulation of BM-MSC differentiation.