淫羊藿苷对环磷酰胺化疗导致小鼠骨髓间充质干细胞成骨分化障碍的保护作用  

Icariin protects against cyclophosphamide-induced obstacle of mouse bone marrow mesenchymal stem cells differentiating into osteoblasts

作　　者：杨志烈 王成龙[2] 赵东峰[2] 常君丽[2] 杨成[1] 杨燕萍[2] 王拥军[2] 

机构地区：[1]滨州医学院中西医结合学院,山东省烟台市264003 [2]上海中医药大学附属龙华医院,上海市200032

出　　处：《中国组织工程研究》2016年第6期777-784,共8页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金面上项目(81373667和81202708);国家重点基础研究发展计划("973"计划)基金资助项目(2010CB530400);上海中医药大学"085"一流学科建设(085ZY1217和085ZY1204);教育部创新团队发展计划(IRT1270);国家中医临床研究基地龙医学者;龙医团队计划项目(LYTD-23);上海市科委"扬帆计划"项目(15YF1412200);上海中医药大学龙华医院自然基金(14YR03)~~

摘　　要：背景:化疗引发骨质疏松已成为严重影响骨骼系统的毒副作用之一,淫羊藿苷具有很强的抗骨质疏松活性,可能对化疗引发的骨质疏松具有保护作用。目的:观察淫羊藿苷对环磷酰胺导致小鼠骨髓间充质干细胞成骨分化障碍的保护作用及机制研究。方法:以MTT法检测结合碱性磷酸酶染色,确定淫羊藿苷对环磷酰胺导致的骨髓间充质干细胞成骨分化障碍的最佳保护剂量。采用RT-PCR法测定淫羊藿苷最佳剂量干预下不同时间点成骨转录特异性因子ALP、OC、Runx2及Wnt/β-catenin信号通路相关基因β-catenin、C-Myc、Cyclin D1 m RNA的表达;Western blot测定最佳剂量情况下淫羊藿苷调节骨髓间充质干细胞成骨特异性转录因子Runx2及Wnt/β-catenin信号通路相关基因Activeβ-catenin、C-Myc、Cyclin D1蛋白的表达。结果与结论:1与正常对照组比较,环磷酰胺组细胞活力及碱性磷酸酶染色减弱,相比于环磷酰胺组,淫羊藿苷各浓度组细胞活力无明显差异;碱性磷酸酶染色显示100μmol/L淫羊藿苷对骨髓间充质干细胞成骨分化障碍的保护作用最佳。2与正常对照组比较,环磷酰胺组降低了成骨指标ALP、OC、Runx2m RNA和Runx2蛋白表达,减弱了Wnt/β-catenin信号通路相关基因β-catenin、Cyclin D1 m RNA及Activeβ-catenin、Cyclin D1、c-myc蛋白的表达水平,增加了Wnt/β-catenin信号通路抑制蛋白DKK1表达;与环磷酰胺组比较,100μmol/L淫羊藿苷能促进各成骨相关指标及Wnt/β-catenin信号通路相关基因的m RNA和蛋白水平,并降低DKK1蛋白表达。3结果表明,环磷酰胺能导致小鼠骨髓间充质干细胞成骨分化障碍,淫羊藿苷对其具有保护作用,且100μmol/L浓度的淫羊藿苷干预效果最佳,而这种保护作用可能与淫羊藿苷激活Wnt/β-catenin信号通路有关。BACKGROUND： Osteoporosis caused by chemotherapy has become one of the serious side effects that impact the skeletal system. Icariin shows a strong anti-osteoporosis activity, which can have protective effect on osteoporosis induced by chemotherapy. OBJECTIVE： To study the protective effect and mechanism of icariin against cyclophosphamide-induced obstacle of mouse bone marrow mesenchymal stem cells differentiating into osteoblasts. METHODS： MTT assay and alkaline phosphatase（ALP） staining were used to determine the optimal protective concentration of icariin against cyclophosphamide-induced obstacle of mouse bone marrow mesenchymal stem cells differentiating into osteoblasts. m RNA expressions of osteoblast-specific transcription factors, OC, ALP, Runx2, and Wnt/β-catenin signaling pathway target genes, β-catenin, C-Myc, cyclin D1, were determined using RT-PCR method at different time after intervention with the optimal concentration of icariin. Expressions of Runx2, β-catenin, c-Myc, cyclin D1 regulated by the optimal concentration of icariin were detected using western blot assay at the protein level. RESULTS AND CONCLUSION： Cell viability and ALP activity decreased significantly in the cyclophosphamide group compared with the control group, but there was no significant difference in cell viability between icariin group and cyclophosphamide group. Icariin at 100 μmol/L showed the best protective effect against cyclophosphamide-induced obstacle of osteogenic differentiation of bone marrow mesenhymal stem cells. Compared with the control group, cyclophosphamide chemotherapy reduced the expressions of ALP, OC, Runx2 at m RNA level and Runx2 at protein level, weakened the expressions of β-catenin, cyclin D1 at m RNA level and active β-catenin, Cyclin D1, c-myc at protein level, and increased the expression of DKK1. Compared with the cyclophosphamide group, 100 μmol/L icariin increased the expression of osteoblast-specific transcription factors and Wnt/β-catenin signaling pathway genes at m RNA a

关 键 词：干细胞 骨髓干细胞 环磷酰胺 骨髓间充质干细胞 成骨分化 淫羊藿苷 保护作用Wnt/β-catenin 国家自然科学基金 

分 类 号：R394.2[医药卫生—医学遗传学]