CAJ | 학술논문

目的探讨淫羊藿甙对骨形态发生蛋白细胞信号通路Smad1、Smad5和Smad4表达的调节作用及促进成骨细胞增殖与分化的机制.方法在体外分别用含淫羊藿甙0、10-9、10-8和10-7 mol/L 的条件培养液干预MC3T3-E1细胞.给药后第1天、2天和3天,分别应用四甲基噻唑蓝法绘制细胞生长曲线并计算细胞群体倍增时间,用速率分光光度法测定碱性磷酸酶含量,用RT-PCR和Western blot 技术检测Smad1、Smad5、Smad4及骨钙素、Runx2、骨形态发生蛋白-2、骨保护素mRNA的表达,于细胞生长的第21天观察钙结节数量.结果淫羊藿甙含量为10-8 mol/L时,MC3T3-E1细胞增殖能力最强,碱性磷酸酶活性和钙结节数量明显增加；淫羊藿甙作用细胞第1天、2天和3天时,10-8 mol/L组细胞Smad1、Smad5和Smad4 mRNA的表达量始终保持较高水平,第3天时10-9 mol/L和10-7 mol/L组表达明显减少；第2天时,10-8 mol/L组细胞Runx2、骨形态发生蛋白-2、骨保护素mRNA和骨钙素、Smadl、Smad5和Smad4蛋白表达明显增加.结论淫羊藿甙通过直接刺激MC3T3-El细胞的骨形态发生蛋白-2、Runx2、Smadl、Smad5和Smad4的表达,且以表达水平同步增高的方式促进其成骨性分化.
목적 탐토음양곽대대골형태발생단백세포신호통로Smad1、Smad5화Smad4표체적조절작용급촉진성골세포증식여분화적궤제.방법 재체외분별용함음양곽대0、10-9、10-8화10-7 mol/L 적조건배양액간예MC3T3-E1세포.급약후제1천、2천화3천,분별응용사갑기새서람법회제세포생장곡선병계산세포군체배증시간,용속솔분광광도법측정감성린산매함량,용RT-PCR화Western blot 기술검측Smad1、Smad5、Smad4급골개소、Runx2、골형태발생단백-2、골보호소mRNA적표체,우세포생장적제21천관찰개결절수량.결과 음양곽대함량위10-8 mol/L시,MC3T3-E1세포증식능력최강,감성린산매활성화개결절수량명현증가；음양곽대작용세포제1천、2천화3천시,10-8 mol/L조세포Smad1、Smad5화Smad4 mRNA적표체량시종보지교고수평,제3천시10-9 mol/L화10-7 mol/L조표체명현감소；제2천시,10-8 mol/L조세포Runx2、골형태발생단백-2、골보호소mRNA화골개소、Smadl、Smad5화Smad4단백표체명현증가.결론 음양곽대통과직접자격MC3T3-El세포적골형태발생단백-2、Runx2、Smadl、Smad5화Smad4적표체,차이표체수평동보증고적방식촉진기성골성분화.
Objective To explore the regulation function of Icariine on the expression of bone morphogenetic protein signaling pathway Smad1,Smad5,Smad4 and to explore the mechanism of promoting MC3T3-E1 cell proliferation and differentiation.Methods MC3T3-E1 cells were treated by 0,10-9,10-8 and 10-7 mol/L Icariine respectively.After stimulated by Icariine 1 d,2 d and 3 d,MTT method and population diploid time were used to observe the cell proliferation,and the cell alkaline phosphatase (ALP) level was assayed.At 21 days later,the alizarin red staining was proceeded.At 1,2 and 3 days later,the RT-PCR was used to detect the mRNA expression level about Smad1,Smad5 and Smad4,and the Western blot was to detect the Smad1,5 and Smad4 protein.At 2 days later,the RT-PCR was used to detect the mRNA expression level about Runx2,BMP-2 and osteoprotegerin (OPG),and the Western blot was used to analyze osteocalcin (OCN) protein level.Results After simulated by Icariine,the proliferation (MTT test),the ALP activity and mineralization of osteoblasts were increased,the cell population diploid was reduced (P＜0.05).At 1,2 and 3days later,the results of RT-PCR showed that Icariine continued increasing the mRNA level of Smad1,5 and Smad4 in 10-8 mol/L.At 2 days later,Smad1,Smad5 and Smad4 mRNA expression were obviously reduced in 0 mol/L group,and At 3 days later,Smad1,Smad5 and Smad4 mRNA expression were obviously reduced in 10-9 and 10-7 mol/L groups.At 2 days later,BMP-2,Runx2 and OPG mRNA were obviously increased in 10-8 mol/L group.The results of Western blot showed that OCN,Smad1,Smad5 and Smad4 protein were obviously up-regulated in 10-8 mol/L group.Conclusion Icariine occurs the expressions of BMP-2,Runx2,Smad1,Smad5 and Smad4 by stimulating the bone morphology of MC3T3-E1 cells directly,and promotes the osteogenic differentiation in the manner of expression level synchronous rising.