补骨脂素对环磷酰胺抑制小鼠骨髓间充质干细胞成骨分化的恢复作用  

Restoration of osteogenic differentiation of bone marrow mesenchymal stem cells in mice inhibited by cyclophosphamide with psoralen

作　　者：王成龙[1] 杨志烈 常君丽 赵永见 赵东峰 戴薇薇[1] 吴宏进[1] 张婕 王利波[1] 谢颖 唐德志 王拥军 杨燕萍 Wang Chenglong;Yang Zhilie;Chang Junli;Zhao Yongjian;Zhao Dongfeng;Dai Weiwei;Wu Hongjin;Zhang Jie;Wang Libo;Xie Ying;Tang Dezhi;Wang Yongjun;Yang Yanping(Central Laboratory for Science and Technology,Longhua Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China;Institute of Spinal Disease,Longhua Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China;Institute of Traditional Chinese Medicine Surgery,Longhua Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China)

机构地区：[1]上海中医药大学附属龙华医院科技中心实验室,上海市200032 [2]上海中医药大学附属龙华医院脊柱病研究所,上海市200032 [3]上海中医药大学附属龙华医院中医外科研究所,上海市200032

出　　处：《中国组织工程研究》2025年第1期16-23,共8页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金青年项目(81202708),项目负责人:王成龙;国家重点研发计划(2018YFC1704300),项目负责人:杨燕萍;国家自然科学基金(81973877,82174408),项目负责人:杨燕萍。

摘　　要：背景:补骨脂素具有很强的抗骨质疏松活性,可能对化疗导致的骨质疏松具有恢复作用。目的:探讨补骨脂素对环磷酰胺抑制小鼠骨髓间充质干细胞成骨分化的恢复作用及机制。方法:分离培养C57BL/6小鼠骨髓间充质干细胞,以MTT法检测补骨脂素对骨髓间充质干细胞活力的影响,以成骨诱导结合碱性磷酸酶染色确定补骨脂素对环磷酰胺抑制骨髓间充质干细胞成骨分化恢复作用的最佳剂量。采用RT-qPCR法检测补骨脂素干预不同时间点成骨分化标志基因Runx2、ALP、Osteocalcin、骨保护素及Wnt/β-catenin信号通路相关基因Wnt1、Wnt4、Wnt10b、β-catenin、c-MYC的mRNA表达;采用Western blot法检测补骨脂素干预不同时间点成骨特异性转录因子Runx2及Wnt/β-catenin信号通路相关基因Activeβ-catenin、DKK1、c-MYC、Cyclin D1的蛋白表达。结果与结论:(1)不同浓度补骨脂素对骨髓间充质干细胞活力没有显著影响;200μmol/L补骨脂素干预后对环磷酰胺诱导骨髓间充质干细胞成骨分化抑制的恢复作用最佳;(2)补骨脂素可以逆转环磷酰胺条件培养基导致的骨髓间充质干细胞成骨标志基因Runx2、ALP、Osteocalcin、骨保护素mRNA表达和Runx2蛋白表达的降低;(3)补骨脂素可以逆转环磷酰胺条件培养基导致的骨髓间充质干细胞Wnt/β-catenin通路相关基因Wnt4、β-catenin、c-MYC mRNA表达和Activeβ-catenin、c-MYC、Cyclin D1蛋白表达的降低以及DKK1蛋白表达的升高;(4)结果表明,环磷酰胺能抑制小鼠骨髓间充质干细胞成骨分化,补骨脂素对其具有恢复作用,且200μmol/L补骨脂素干预效果最佳,而这种保护作用可能与补骨脂素激活Wnt4/β-catenin信号通路有关。BACKGROUND:Psoralen has a strong anti-osteoporotic activity and may have a restorative effect on chemotherapy-induced osteoporosis.OBJECTIVE:To explore the restorative effect of psoralen on the osteogenic differentiation of bone marrow mesenchymal stem cells in mice inhibited by cyclophosphamide and its mechanism.METHODS:C57BL/6 mouse bone marrow mesenchymal stem cells were isolated and cultured.Effect of psoralen on viability of bone marrow mesenchymal stem cells was detected by MTT assay.Osteogenic induction combined with alkaline phosphatase staining was used to determine the optimal dose of psoralen to restore the osteogenic differentiation of bone marrow mesenchymal stem cells inhibited by cyclophosphamide.The mRNA expression levels of Runx2,alkaline phosphatase,Osteocalcin,osteoprotegerin,and Wnt/β-catenin signaling pathway-related genes Wnt1,Wnt4,Wnt10b,β-catenin,and c-MYC were measured by RT-qPCR at different time points under the intervention with psoralen.The protein expression of osteogenic specific transcription factor Runx2and Wnt/β-catenin signaling pathway related genes Activeβ-catenin,DKK1,c-MYC,and Cyclin D1 was determined by western blot assay at different time points under the intervention with psoralen.RESULTS AND CONCLUSION:(1)There was no significant effect of different concentrations of psoralen on the viability of bone marrow mesenchymal stem cells.The best recovery of the inhibition of osteogenic differentiation of bone marrow mesenchymal stem cells caused by cyclophosphamide was under the intervention of psoralen at a concentration of 200μmol/L.(2)Psoralen reversed the reduction in osteogenic differentiation marker genes Runx2,alkaline phosphatase,Osteocalcin and osteoprotegerin mRNA expression and Runx2 protein expression in bone marrow mesenchymal stem cells caused by cyclophosphamide conditioned medium.(3)Psoralen reversed the decrease in Wnt/β-catenin pathway-related genes Wnt4,β-catenin,c-MYC mRNA and Activeβ-catenin,c-MYC,and Cyclin D1 protein expression and the increase in

关 键 词：环磷酰胺 骨髓间充质干细胞 成骨分化 补骨脂素 Wnt4 Β-CATENIN 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学] R318