淫羊藿甙对MC3T3-E1细胞Smad1，5作用的实验研究

The experiment study of the effects of icariin on Smadl, 5 in MC3T3-E1 cell in vitro

目的检测淫羊藿甙（icariin，ICA）对MC3T3-E1中Smad1，5mRNA及蛋白的影响。方法DMEM高糖、胎牛血清培养下的MC3T3-E1细胞按ICA刺激浓度0、10、40及80ng／ml分为四组，各组接种于6孔板中，接种细胞数目约为3×10^5个／孔，分别于给药后24、48及72h，用半定量RT-PCR技术测定Smadl，5mRNA的量，以Western blot评价Smad1，5蛋白的量。并于给药72h后用免疫组织化学定位Smad1，5蛋白的表达。采用SPSS 13．0软件进行统计学分析。结果RT-PCR显示ICA刺激24h后，0、10、40、80ng／ml各组Smad1，5mRNA量的表达无统计学差异；刺激48h后，0ng／ml组Smad1 mRNA表达量检出极少，Smad5 mRNA未检出；至72h时，0ng／ml组二者均未检出，而10、40、80ng／ml各组在刺激48、72h时，Smad1，5mRNA保持较高水平，各组Smad1，5mRNA的表达量较0ng／ml组具有统计学意义。Western blot蛋白印迹显示10、40、80ng／ml各组的Smadl蛋白表达于不同时间点较0ng／ml组明显增加，0ng／ml组仅于刺激72h后少量检出。Smad5蛋白表达除0ng／ml组外，于10、40、80ng／ml各组在不同时间点均有检出，较0ng／ml组具有统计学意义。免疫组织化学显示10、40、80ng／ml组，于胞质及核内Smad1，5蛋白的表达较0ng／m组均增多。结论淫羊藿甙可能通过上调Smad1，5mRNA及蛋白的表达来促进成骨细胞分化。

Objective To explore the effect of ieariin on the expression of Smadl, 5 mRNA and protein Smadl, 5 in MC3T3-E1 cells in vitro. Methods According to the stimulus concentrations （0, 10, 40 and 80 ng/ml ） of icariin, the MC3T3-E1 cells were divided into four groups. After stimulated by icariin 24, 48 and 72 h, the RT-PCR was used to detect the mRNA expression level about Smadl, 5. Western blot technique was applied to exsplore the Smadl, 5 protein expression. The immunohistochemistry was engaged to eomfirm and to localize the expression of Smadl, 5 protein in MC3T3-E1 cells. All data were treated with One-way Anova analysis and Dunnett-t test in SPSS 13.0 software. Results After stimulated by inariin of 48 h and 72 h, both Smadl, 5 mRNA increased continuously by time in 10, 40 and 80 ng/ml, the difference were statistical significance. But in 0 ng/ml group, Smadl, 5 mRNA was no obviously increased. At 72 h, samdl, 5 mRNA were not observed expression in 0 ng/ml groups. Western blot demonstrated that Smadl and protein under the stimulation of different dose of icariin expressed much more than 0 ng/ml groups at different times. Western blot shown that Smadl, 5 protein level were higher in 10, 40 and 80 ng/ml, than that in 0 ng/ml group at 24, 48 or 72 h, the difference was statistical significance. Immunohistochemistry displayed that in 10, 40 and 80 ng/ml groups, the expression both of the Smadl, 5 proteins were strongly postire in cytoplasm and nuclei, but was mildly positve in 0 ng/ml group. Conclusion Icariin is able to upregulate the expression level of Smadl, 5 mRNA and protein in vitro.