淫羊藿苷对衰老骨髓间充质干细胞成骨的影响  

Effects of icariin on osteogenesis of aging bone marrow mesenchymal stem cells

作　　者：蔡叶 王明飞[1] 张磊[1] 王健[2] 石继祥[1] 仇建军 刘铖祎[1] 周旭 CAI Ye;WANG Mingfei;ZHANG Lei;WANG Jian;SHI Jixiang;QIU Jianjun;LIU Chenyi;ZHOU Xu(Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200062;Pudong New Area People’s Hospital,Shanghai 201200)

机构地区：[1]上海中医药大学附属普陀医院,上海200062 [2]上海市浦东新区人民医院,上海201200

出　　处：《中国骨质疏松杂志》2023年第2期162-167,共6页Chinese Journal of Osteoporosis

基　　金：上海市卫生健康委员会科研课题计划(202040151);上海市普陀区中心医院、上海中医药大学附属普陀医院临床匠才计划(2022-RCJC-08)。

摘　　要：目的探究淫羊藿苷(icarrin,Ica)对D-半乳糖(D-gal)诱导的衰老骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨的影响及机制。方法通过全骨髓贴壁法提取BMSCs,采用流式细胞术及成骨、成脂诱导分化鉴定细胞。通过CCK8法检测D-gal及Ica处理BMSCs的最适浓度,用D-gal诱导BMSCs衰老,药物干预48 h;6SA-β-半乳糖苷酶染色检测细胞衰老情况;碱性磷酸酶试剂检上清液ALP水平;RT-PCR和Western Blot分别检测BMSCs中衰老相关P16、P53、P21及成骨相关Foxo3a、β-catenin的蛋白表达。结果BMSCs表面标志物CD44和CD90表达大于95%;CD34、CD45表达小于5%,且能被定向诱导分化出钙结节及脂滴,鉴定细胞为BMSCs。CCK8检测出30 mg/mL为D-gal的最适浓度,10-8、10-10mol/L为Ica最适浓度。Ica能减少衰老BMSCs中P16、P53、P21 mRNA的表达;降低Foxo3a mRNA和蛋白表达,增加β-catenin mRNA和蛋白的表达,差异具有统计学意义(P<0.05)。结论D-gal能够诱导构建出衰老BMSCs模型;Ica增加了衰老BMSCs上清液中的ALP含量、降低了衰老BMSCs中P53的表达水平,可能通过调节成骨相关信号通路Foxo3a/β-catenin表达,起到对衰老BMSCs成骨分化能力的改善作用。Objective To investigate the effect of Icarrin(Ica)on Bone marrow mesenchymal stem cells(BMSCs)induced by D-galactose(D-Gal)and its mechanism.Methods BMSCs were extracted by whole bone marrow adherent method,and differentiated by flow cytometry and osteogenic and adipogenic induction.CCK8 method was used to detect the optimal concentration of BMSCs treated with d-gal and Ica.D-gal was used to induce aging of BMSCs,and drug intervention was performed for 48 h.SA-β-galactosidase staining was used to detect cell senescence.ALP level of supernatant was detected by alkaline phosphatase reagent.The expression of aging-related p16,p53,p21 and osteogenic Foxo3a andβ-catenin proteins in BMSCs were detected by RT-PCR and Western Blot respectively.Results The expression of CD44 and CD90 on BMSCs was more than 95%.The expression of CD34 and CD45 was less than 5%,and they could be induced to differentiate into calcium nodules and lipid droplets.The cells were identified as BMSCs.CCK8 detected that 30 mg/mL was the optimal concentration of D-Gal,and 10-8and 10-10mol/L were the optimal concentration of Ica.Ica could reduce the expression of P16,P53 and P21 mRNA in aging BMSCs.The expression of Foxo3a mRNA and protein was decreased,and the expression ofβ-catenin mRNA and protein was increased,the difference was statistically significant(P<0.05).Conclusion D-Gal can induce the establishment of aging BMSCs model.Ica could increase the content of ALP in supernatant of aging BMSCs.It can reduce the expression levels of P53 in aging BMSCs,and may improve the osteogenic differentiation ability of aging BMSCs by regulating the expression of Foxo3a/β-catenin.

关 键 词：中医中药 老年性骨质疏松症 骨髓间充质干细胞 淫羊藿苷 D-半乳糖 

分 类 号：R285.5[医药卫生—中药学]