CAJ | 학술논문

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.
목적 검측음양곽대(icariin,ICA)대MC3T3-EI중Smad1,5 mRNA급단백적영향.방법 DMEM고당、태우혈청배양하적MC3T3-E1세포안ICA자격농도0、10、40급80 ng/ml분위사조,각조접충우6공판중,접충세포수목약위3×105개/공,분별우급약후24、48급72 h,용반정량RT-PCR기술측정Smad1,5 mRNA적량,이Western blot평개Smad1,5단백적량.병우급약72h후용면역조직화학정위Smad1,5단백적표체.채용SPSS 13.0연건진행통계학분석.결과 RT-PCR현시ICA자격24 h후,0、10、40、80 ng/ml각조Smad1.5 mRNA량적표체무통계학차이;자격48 h후,0 ng/ml조Smad1mRNA표체량검출겁소,Smad5 mRNA미검출;지72 h시,0 ng/ml조이자균미검출,이10、40、80 ng/ml각조재자격48、72 h시,Smad1,5 mRNA보지교고수평,각조Smad1,5 mRNA적표체량교0 ng/ml조구유통계학의의.Westem blot단백인적현시10、40、80 ng/ml각조적Smad1단백표체우불동시간점교0ng/ml조명현증가,0 ng/ml조부우자격72 h후소량검출.Smad5단백표체제0 ng/ml조외,우10、40、80ng/ml각조재불동시간점균유검출,교0 ng/ml조구유통계학의의.면역조직화학현시10、40、80 ng/ml조,우포질급핵내Smad1,5단백적표체교0 ng/m조균증다.결론 음양곽대가능통과상조Smad1,5mRNA급단백적표체래촉진성골세포분화.
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.