左归丸对骨髓间充质干细胞成骨分化相关基因甲基化的影响  

Influence of Zuogui Wan(左归丸) on Methylation of Osteogenic Differentiation Related Gene in Bone Marrow Mesenchymal Stem Cell

作　　者：杨锋[1,2] 潘乐[2] 马秋涛 张涛[2] 杨利学[1,2] 李彦民[1] 

机构地区：[1]陕西中医学院附属医院、陕西省咸阳市渭阳西路副2号,712000 [2]陕西中医学院第一临床医学院

出　　处：《中医杂志》2015年第15期1326-1329,共4页Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金(81102610,81473711);陕西省中医管理局2013~2015年度中医药科学技术研究课题(13-JC014)

摘　　要：目的研究左归丸对骨髓间充质干细胞(BMSCs)向成骨分化的表观遗传学机制。方法 30只SD大鼠给予左归丸溶液(浓度为472.5 g/L)灌胃,每次1 ml,每日2次,连续灌胃3天后经腹主动脉采血,制备左归丸含药血清。无菌条件下取出SD大鼠双侧股骨和胫骨,收集骨髓冲洗液,贴壁法培养传代所得BMSCs。取2代BMSCs以5×104/cm2密度接种于培养皿,分为3组,对照组常规培养;成骨诱导组用10%空白血清加成骨诱导剂培养;左归丸组用10%左归丸含药血清加成骨诱导剂培养。干预14天后采用RT-PCR及甲基化特异性PCR检测法进行Runx2、Osterix、OC及β-catenin基因表达和甲基化状态检测。结果左归丸组Runx2、Osterix、OC、β-catenin的相对表达量较对照组、成骨诱导组均上调(P<0.05)。左归丸组Runx2、Osterix、OC、β-catenin基因甲基化率均低于对照组、成骨诱导组(P<0.05)。结论左归丸含药血清可通过去甲基化的表观遗传调控机制促进BMSCs向成骨细胞分化。Objective To observe the epigenetic mechanism of Zuogui Wan （左归丸） in osteogenic differentiation of bone marrow mesenchymal stem ceils （BMSCs）. Methods Thirty SD rats were given 1 ml Zuogui Wan solution （472. 5 g/L） by gavage twice a day for continuous 3 days. Afterwards, blood samples were collected and Zuogui Wan serum samples were obtained. Bilateral femur and tibia of SD rats were obtained in asepsis and irrigation fluid of bone marrow was collected. BMSCs were incubated and passaged by adherent method. Second generation of BMSCs were inoculated in the density of 5 × 104/cm2 in culture dishes. They were divided into 3 groups, Control group was conventional cultured. Osteogenic induction group was cultured by 10% blank serum with osteogenic induce supple- ments. Zuogui Wan group was cultured by 10% Zuogui Wan serum with osteogenic induce supplements. After 14 days, gene expression and methylation of Runx2, Osterix and β-catenin were detected by RT-PCR and methylation specific PCR. Results Expressions of Runx2, Osterix, OC and β-catenin were higher in Zuogui Wan group than those in control and osteogenic induction groups （P 〈 0. 05 ）. Methylations of Runx2, Osterix, OC and β-catenin were lower in Zuogui IVan group than those in control and osteogenic induction groups （P 〈 0. 05 ）. Conclusion Zuogui Wan serum can promote BMSCs differentiating to osteoblasts by epigenetic mechanism of demethylation.

关 键 词：左归丸 骨髓间充质干细胞 成骨分化 基因甲基化 

分 类 号：R285[医药卫生—中药学]