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Purpose: the research icariine (icariin, ICA) on cultured osteoblast-like cells MC3T3-E1 of Smad1, 5 mRNA and protein in the molecular mechanisms of ICA stimulate osteoblast growth and differentiation, ICA clinical The application provides evidence of cell and molecular biology. : DMEM high glucose, fetal bovine serum cultured MC3T3-E1 cells according to the ICA to stimulate the concentration of 0,10,40 and the 80ng/ml divided into four groups, each group seeded in 6-well plates, inoculated cells approximately 3 × 10 5 / well, respectively, after administration of 24, 48 and 72 h, the amount of each concentration group were 3 hole Smad1, Smad5 and actin mRNA Determination of semi-quantitative RT-PCR technology, by Western blot Western blot method corresponding to the 3-hole Evaluation of Smad1, Smad5 pSmads protein, relatively actin protein expression. MC3T3-E1 cells were seeded in the number of cells of approximately 1 × 10 ~ 5 cells / well in 6-well plates, cells seeded growth, administered 72h after cells Immunohistochemical localization and validation of Smad1, Smad5 protein expression by immunohistochemistry fluorescent the technical positioning pSmads protein expression. Relevant experimental data using one-way analysis of variance (One-way Anova) and LSD-t test was used for statistical analysis SPSS13.0 software. Results: Semi-quantitative RT-PCR show 24hrs, ICA each concentration group of Smad1, Smad5 mRNA expression levels of statistical significance, P = gt; 0.05 (of Smad1: F = 0.075, P = 0.972; Smad5: F = 1.227, P = 0.362). 48,72 hrs ICA10ng / m, 40ng/ml, 80ng/ml concentration group the Smad1, Smad5mRNA expression level over 0ng/ml group were significantly increased, P = lt; 0.05 was statistically significant (48hrs: Smad1, F = 342.964, P = 0.000; Smad5, F = 395.542, P = 0.000; 72hrs: Smad1, F = 144.120, P = 0.000; Smad5, F = 100.245, P = 0.000). To 72hrs ICA10, 40, 80 ng / ml of each group 10ng/ml group of Smad1, Smad5 mRNA upregulation of the role of the most significant (P = lt; 0.05). Western blot Western blot 24,48 hrs Smad1 protein expression level of the ICA each concentration group was not statistically significant, P value gt; 0.05 (24hrs: F = 0.797, P = 0.529; 48hrs: F = $ 258.3, P = 0.126), 72hrs when ICA10ng/ml group, 40ng/ml group and 80ng/ml group Smad1 protein expression levels over 0ng/ml group significantly increased P value lt; 0.05 (F = 17.015, P = 0.001), a statistically significant; Smad5 protein expression in ICA10 40,80 ng / ml, respectively, compared with 0ng/ml group raised 24,48,72 hrs, which 48hrs, 3 days after the 3rd day of exercise express significant incremental P value lt; 0.05 was statistically significant (24hrs: F = 3.272, P = 0.080; 48hrs: F = 15.929, P = 0.001; 3 days after the 3rd day of exercise: F = 28.362, P = 0.000); the pSmad1, 5 protein the in ICA10ng / m 40ng/ml, 80ng/ml each concentration group 24hrs ( F = 1.462, P = 0.296) of than the 0ng/ml P value gt; 0.05, not statistically significant (F = 1.462, P = 0.296), 48, 72 hrs 0ng/ml upregulation of P lt; 0.05 statistically significant (48hrs: F = 6.407, P = 0.016; 72hrs: F = 4.631, P = 0.037). ICA effective stimulation group compared with the control group Smad5 protein expression in 24hr when that difference of Smad1 expression differences in 72hrs expression differences relative to 48hrs Smad1 mRNA in Smad5 mRNA in terms of the expression of the protein is not parallel to the Smad1, Smad5 gene expression, suggesting that the regulation of the ICA Smad1, the 5 signal post-transcriptional level; activated Smad1, 5 protein differences 48hrs later, the difference late in the Smad5 protein before Smad1 protein, suggesting that Smad5 protein may in the the two Smads signal system express priority. To 72hrs ICA10, 40, 80 ng / ml of each group 10ng/ml group of Smad1, Smad5 pSmad1, the 5 protein Upregulation of the most significant (P = lt; 0.05). Immunohistochemistry staining revealed 10,40,80 ng / ml group Smad1, 5 protein expression than 0ng / m group, not only in the cytoplasm of a significant increase in nucleus Smad1, 5 protein corresponding increase in the prompt of Smad1 protein increase as a the cytoplasm messenger to the nuclear translocation. Immunofluorescence display ICA can be significant with the increase in the content of the cell slurry and nuclear pSmad1 protein of, indirectly prompted ICA be able to increase the Smad1, 5 of 5 protein of the activated form pSmad1, 5 of 5 proteins of the content, and further confirmed ICA having conduction and BMP-2 with similar extracellular passage that BMP2-Smad1, 5 signal pathways. Conclusion: ICA may be raised Smad1, 5mRNA and protein expression to promote osteoblast differentiation.
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