Thioacetamide Induces Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Osteoclasts

Objective To investigate the effects of thioacetamide(TAA)on the proliferation,apoptosis and differentiation on BMSCs by isolation,culture and identification of BMSCs in vitro,and to investigate the differentiation of BMSCs into osteoclast-like cells induced by TAA in vitro,To analyze the changes of osteoclast-like morphology,protein expression and the expression of bone metabolism markers during the induction process,to provide technical support and experimental evidence for the establishment of an animal model of bone and joint degenerative diseases.To establish an antiosteoporosis drug screening model and to study its mechanism to provide a research platform in vitro for the development and research of prophylactic and / or therapeutic drugs.Methods 1.Isolation and culture of rat bone marrow mesenchymal stem cells(BMSCs)sterilely in vitro and detect surface molecular of the cells.2.The optimal concentration of thioacetamide-induced BMSCs differentiation was screened by CCK-8 assay.The effect of TAA on cell division of BMSCs was detected by CFSE.3.Flow cytometry was used to detect the effect of TAA on apoptosis and cell cycle;Western Blot was used to detect the expression of apoptosis related protein.4.BMSCs differentiated osteoclasts were specifically morphologically stained by Tartrate-resistant acid phosphatase(TRAP)staining.5.Western Blot assay was used to verify the effects of TAA on osteoclast-specific protein expression of BMSCs.6.Immunofluorescence assay was used to verify the effects of TAA on osteoclastspecific protein expression of BMSCs.7.Intracellular calcium of BMSCs treated TAA was detected by Fluo-3 AM fluorescence.8.Bone metabolism markers of BMSCs treated with TAA were detected by Electrochemiluminescence.Results 1.Rat bone marrow mesenchymal stem cells(BMSCs)expressed standard surface markers of stem cell markers were isolated and cultured in vitro successfully which can be used in this experimental study.2.The results of CCK-8 showed that TAA could inhibited the proliferation of bone marrow stromal cells in a time-and dose-dependent manner.The results of CFSE showed that TAA could inhibit the division of bone marrow stromal cells.3.Flow cytometry results showed that: the apoptosis rat of bone marrow stromal cells treated with TAA significantly increased,arrested cells in the small phase significantly increased.4.The number of TRAP-positive cells in RANKL combined with M-CSF group,TAA 0.5mg / m L group and TAA 1mg / m L group were significantly increased compared with the control group after 3 and 7 days of induction respectively(P<0.05),and more positive cells were obtained in TAA group than RANKL combined with M-CSF induction group.5.Western Blot results showed that compared with the control group,the osteoclastspecific protein of TRAP and cathepsin k protein in the group of RANKL combined with M-CSF,TAA 0.5mg / m L group and TAA 1mg / m L group respectively after 3 days and 7 days of induction were upregulated.6.Immunofluorescence results showed that compared with the control group,the osteoclast-specific proteins of TRAP and Cathepsin K protein in the group RANKL combined with M-CSF group,TAA 0.5mg / m L group and TAA 1mg / m L group were decreased after 3 days and 7 days induction were upregulated.7.Fluo-3 AM results show that: compared with the control group,intracellular calcium of BMSCs treated with TAA were increased.8.The results of bone metabolic markers showed that the levels of PTH and N-terminal osteocalcin were upregulated and the expression of 1-25-hydroxyvitamin D was downregulated after 3 days and 7 days respectively.Conclusion 1.BMSCs were cultured in vitro in various sizes,with various morphologies,such as astrocytes and fusiform shape.The BMSCs cells positive for surface of CD29 and CD90 and negative for hematopoietic markers CD45 and CD11b/c.2.The proliferation of BMSCs could be inhibited by TAA,BMSCs could be induced to apoptosis and the cells were arrested in S phase.3.The BMSCs cells induced by TAA differentiated into osteoclast-like cells and positive for TRAP staining.4.The expression of osteoclast-specific protein of TRAP and Cathepsin K were upregulated by TAA.5.The expression of bone resorption markers and bone turnover rate were enhanced by TAA.