| Objective:To explore the effect of icariin on the expression of Smadl,5 mRNA and protein Smadl,5 in preosteoblast MC3T3-E1 cells in vitro.Methods:MC3T3-E1 cells were divided into four groups according to stimulus concentrations of icariin,those were 0,10,40,80ng/ml groups.Each dose group cells were planted in 6-well plate and the plantation number of MC3T3-E1 cells was 3×10~5 in each well.Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to find out the mRNA expression level about Smad1,Smad5 and actin.Western blot technique was adopted to detect the protein expression level of them.MC3T3-E1 cells were cultured in 6-well plate which was put in with cover glasses preliminarily and the initial cellular plantation number was 1×10~5 in each well.After 72hrs cultured,expression of Smadl,5 or pSmadl protein was localized and confirmed by immunohistochemistry staining or immunofluorescence.The relevant data for RT-PCR and Western blot results were dealt with SPSS 13.0 software.ResuIts:RT-PCR exhibited that both Smadl mRNA and Smad5 mRNA expressed continuously as time went ahead in 10ng/ml groups and they also expressed in 40ng/ml groups or 80ng/ml groups when cells were cultured till 48hrs and 72hrs.But the mRNA expression of Smadl were found a little in 0ng/ml control groups at 48hrs but Smad5 mRNA was not detected expression at the time.At 72hrs,both of them were not observed expression in 0ng/ml groups.The mean net optical density ratios between Smadl or Smad5 mRNA and actin mRNA were shown statistical significance(48hrs:Smadl,F=342.964,P=0.000;Smad5,F=395.542,P=0.000and72hrs: Smadl,F=144.120,P=0.000;Smad5,F=100.245,P=0.000) between the effective stimulus dose groups of icariin and 0ng/ml groups at 48hrs or 72hrs.At 24hrs, whether Smadl or Smad5 expression level,the variances among all groups had no statistical significance(Smadl:F=0.075,P=0.972;Smad5:F=1.227,P=0.362).Western blot demonstrated that the mean net optical density ratios between Smadl protein and actin protein were shown statistical significance(72hrs:F=17.015,P=0.001)between any one of the three effective stimulus dose groups and 0ng/ml groups at 72hrs.The mean net optical density ratios of both Smad5 protein and actin protein were shown statistical significance(24hrs:F=3.272,P=0.080;48hrs:F=15.929,P=0.001;72hrs: F=28.362,P=0.000)at 24,48,72hrs.Phosphorylated protein of Smadl,5 was detected to have statistical significance at 48,72hrs(48hrs:F=6.407,P=0.016;72hrs:F=4.631, P=0.037).Contrasted to difference emergence of Smadl or Smad5 mRNA after 48hrs,disparity of protein Smadl or Smad5 between three effective ICA stimulus dose groups and 0ng/ml groups emerged respectively after 24hrs or at 72hrs. Non-equal pace between mRNA and prtein level for Smadl and Smad5 may show existence of some kind post-trancriptional regulating mechanism for Smadl,5 signal. Variance of pSmadl,5 protein emerged after 48hrs,earlier to Smadl,later than Smad5,and the phenomenon could show protein Smad5 probabily has the expressed prior status in secondary Smads signal system.Immunohistochemistry staining displayed that 10,40 and 80ng/ml groups could promote expression of Smadl,5 proteins in cytoplasm than 0ng/ml group,furthermore,Smadl,5 proteins were observed increasing in cellular nuclei as well.Immunofluorescence demonstrated that ICA can effectively increase the quantity of protein pSmadl in cytoplasm and nuclei in MC3T3-E1 cells,which indirectively showed that ICA could up-regulate content of pSmadl,5(active pattern of protein Smadl,5) in cytoplasm and nuclei.ConcIusion:Icariin is able to up-regulate the expression level of Smadl,5 mRNA and protein Smadl,5 through which to be probably stimulate MC3T3-E1 cell differentiation. |
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