| Objective The aim of this study is to investigate the effect of MC3T3-El cells on proliferation,apoptosis,cell cycle,mineralization and bone metabolism related factors after exposed to TDF,and to explore mechanism of TDF related bone metabolic abnormalities.Methods 1.The effect of MC3T3-El cells on proliferation after exposed to TDF detected by CCK8.Cultivate MC3T3-E1 cells in vitro,divided into blank control group,DMSO treatment group,TDF treatment group,CCK8 detect proliferation after disturbed 24 h and 48 h;2.The effect of MC3T3-El cells on apoptosis after exposed to TDF detected by flow cytometry.Cultivate MC3T3-E1 cells in vitro,divided into blank control group,DMSO treatment group,TDF treatment group,ANNEXIN V/7AAD dye,flow cytometry detect apoptosis after 24 h and 48 h exposed TDF.3.The effect of MC3T3-El cells on apoptosis after exposed to TDF detected by flow cytometry.Cultivate MC3T3-E1 cells in vitro,divided into blank control group,DMSO treatment group and TDF treatment group,7AAD dye,flow cytometry detect DNA content after 24 h exposed TDF.4.The effect of MC3T3-E1 cells on transcriptional level of bone metabolism related factors(runx2,rankl,opg)after exposed to TDF detected by real-time PCR.Cultivate MC3T3-E1 cells in vitro,divided into DMSO control group and TDF treatment group,extract the total RNA after 5 days TDF exposed,detect transcriptional level of bone metabolism related factors(runx2,rankl,opg)by real-time PCR.5.The effect of MC3T3-E1 cells on translational level of bone metabolism related factors(runx2,rankl,opg)after exposed to TDF detected by Western Blot.Cultivate MC3T3-E1 cells in vitro,divided into DMSO control group and TDF treatment group,extract the total protein after 7 days TDF exposed,detect translational level of bone metabolism related factors(runx2,rankl,opg)by Western Blot.6.The effect of MC3T3-E1 cells on mineralization after exposed to TDF detected by Alizarin red staining testing.Cultivate MC3T3-E1 cells in vitro,divided into NC group(common culture),0n M(excluding TDF inducing culture),0.1 ‰ DMSO group,the three TDF treatment group(concentration of TDF are 50nmol/L,500nmol/L,50000nmol/L respectively),after 21 days exposed TDF,Alizarin red S staining detect mineralization.Results 1.The effect of MC3T3-El cells on proliferation after exposed to TDF: the test results show that,compared to DMSO control group,after 24 h exposed to TDF,three treatment groups of 50nmol/L,500nmol/L,5000nmol/L,have no inhibition in proliferation;after 48 h exposed to TDF,two treatment groups of 50nmol/L,500nmol/L,have no inhibition in proliferation.The results showed that different concentrations of TDF have different effects on proliferation.At the condition of the clinical blood drug concentration(about 500nmol/L),TDF have no effect on the cell proliferation inhibition.2.The effect of MC3T3-El cells on apoptosis after exposed to TDF.According to the result of TDF for cell proliferation inhibition rate,we choose four TDF concentrations(50nmol/L,500nmol/L,5000nmol/L,50000nmol/L),exposed TDF 24 h and 48 h to detect the apoptosis rate.The results showed that TDF had no effect on apoptosis rate of MC3T3-E1.3.The effect of MC3T3-El cells on apoptosis after exposed to TDF.The results showed that 500nmol/L and 5000nmol/L group had no effect on cell cycle,but 50000nmol/L group,the number of G2 phase cell increase,and the number of G1 phase cell decrease,suggesting that the cell cycle retardation happened in G2 phase.4.The effect of MC3T3-E1 cells on transcriptional level of bone metabolism related factors(runx2,rankl,opg).The transcriptional levels of bone metabolism related factors(runx2,rankl and opg),TDF treatment group drop compared the DMSO control group.5.The effect of MC3T3-E1 cells on translational level of bone metabolism related factors(runx2,rankl,opg).The translational levels of bone metabolism related factors(runx2,rankl and opg),TDF treatment group drop compared the DMSO control group.6.The effect of MC3T3-E1 cells on mineralization.Results show that compared with control group 0.1 ‰ DMSO,the mineralization of 500nmol/L group decreased significantly,and 50?mol/L group even without calcium nodule formation.Conclusion 1.The effect of proliferation are large different at different time and different concentrations of TDF,and in the clinical blood drug concentration(about 500nmol/L),there is no effect on cell proliferation inhibition;2.TDF had no effect on apoptosis rate of MC3T3-E1;3.TDF of 50000nmol/L concentration block cell in G2 phase,and in clinical drug concentration(about 500nmol/L)TDF had no effect on cell cycle;4.TDF osteogenesis related metabolic factor runx2,rankl and opg transcription and translation,in the clinical blood drug concentration(about 500nmol/L)TDF had no effect on cell cycle;5.TDF inhibited calcium nodule formation of MC3T3-E1 cell;6.Under the influence of TDF,calcium nodule formation is inhibited,the expression levels of osteogenesis related metabolic factor(runx2,rankl and opg)drop,which can lead to disfunction of bone metabolism,and provide basic research for further research in the mechanism of TDF related bone loss and osteoporosis. |
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