淫羊藿甙对MC3T3-E1细胞Smad1,5作用的实验研究

摘要

Objective To explore the effect of icariin on the expression of Smad1, 5 mRNA and protein Smad3, 5 in MC3T3-E1 cells in vitro. Methods According to the stimulus concentrations (0, 30, 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 Smad1, 5. Western blot technique was applied to exsplore the Smad1, 5 protein expression. The immunohistochemistry was engaged to comfirm and to localize the expression of Smad1, 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 Smad1, 5 mRNA increased continuously by time in 10, 40 and 80 ng/ml, the difference were statistical significance. But in 0 ng/ml group, Smad1, 5 mRNA was no obviously increased. At 72 h, samd1, 5 mRNA were not observed expression in 0 ng/ml groups. Western blot demonstrated that Smad1 and protein under the stimulation of different dose of icariin expressed much more than 0 ng/ml groups at differ-ent times. Western blot shown that Smadl, 5 protein level were higher in 30, 40 and 80 ng/ml, than that in 0 ng/ml group at 24, 48 or 72 h, the difference was statistical significance. Immunohistochemistry dis-played that in 10, 40 and 80 ng/ml groups, the expression both of the Smad1, 5 proteins were strongly pos-tive in cytoplasm and nuclei, but was mildly positve in 0 ng/ml group. Conclusion Icariin is able to up-regulate the expression level of Smad1, 5 mRNA and protein in vitro.%目的 检测淫羊藿甙(icariin,ICA)对MC3T3-EI中Smad1,5 mRNA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80 ng/ml分为四组,各组接种于6孔板中,接种细胞数目约为3×105个/孔,分别于给药后24、48及72 h,用半定量RT-PCR技术测定Smad1,5 mRNA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24 h后,0、10、40、80 ng/ml各组Smad1.5 mRNA量的表达无统计学差异;刺激48 h后,0 ng/ml组Smad1mRNA表达量检出极少,Smad5 mRNA未检出;至72 h时,0 ng/ml组二者均未检出,而10、40、80 ng/ml各组在刺激48、72 h时,Smad1,5 mRNA保持较高水平,各组Smad1,5 mRNA的表达量较0 ng/ml组具有统计学意义.Westem blot蛋白印迹显示10、40、80 ng/ml各组的Smad1蛋白表达于不同时间点较0ng/ml组明显增加,0 ng/ml组仅于刺激72 h后少量检出.Smad5蛋白表达除0 ng/ml组外,于10、40、80ng/ml各组在不同时间点均有检出,较0 ng/ml组具有统计学意义.免疫组织化学显示10、40、80 ng/ml组,于胞质及核内Smad1,5蛋白的表达较0 ng/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.