全反式维甲酸增强BMP9诱导的间充质干细胞成骨分化.doc

全反式维甲酸增强骨形态蛋白9诱导间充质干细胞成骨分化作用研究 黄 帆1，刘映孜2，杨秋珺2,周龙洋2，周岐新2，罗进勇3，何百成2,邓忠良 （400010 重庆，重庆医科大学附属第二医院骨科1；400016 重庆，重庆医科大学药理教研室，重庆医科大学检验学院2） [摘要] 目的 研究全反式维甲酸（ATRA）对骨形态蛋白9（BMP9）诱导间充质干细胞成骨分化的影响及可能机制 方法 利用组织化学染色检测处理后第5、7天碱性磷酸酶活性，Western blot方法检测第9、11天骨桥素蛋白表达水平及Smad1/5/8磷酸化水平，茜素红染色检测第14、20天钙盐沉积，萤光素酶报告质粒检测BMPR-Smad信号通路的活化程度。结果 ATRA及BMP9均诱导C3H10T1/2细胞碱性磷酸酶活性升高，但ATRA合并BMP9组碱性磷酸酶活性明显强于ATRA或BMP9组；Western blot结果显示，BMP9合并ATRA组骨桥素蛋白表达水平高于BMP9组；BMP9合并ATRA组钙盐沉积染色也明显于BMP9组。BMP9合并ATRA组BMPR-Smad报告质粒萤光素酶活性明显强于BMP9组（P0.01），Samd1/5/8磷酸化水平也呈相同变化。结论 ATRA能增强BMP9诱导的间充质干细胞成骨分化，其机制可能与ATRA促进Smads信号转录活性有关。 [关键词] 全反式维甲酸；骨形态蛋白9；干细胞；Smad信号 [中图法分类号] R681.1 [文献标志码] A [通信作者] 邓忠良，电话：（023 E-mail：deng7586@ Research on the effect of all-trans retinoic acid enhance the osteogenic differentiation induced by BMP9 in mesenchymal stem cells Huang Fan1, Liu Yingzi2, Yang Qiujun2, Zhou Longyang2, Zhou Qixin2, Luo Jinyong2, He Baicheng2, Deng Zhongliang1 （1Department of Orthopaedics, the Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010；2Department of Pharmacology, Chongqing Medical University, Chongqing， 400016, China） [Abstract] Objective To investigate the effect of all-trans retinoic acid (ATRA) on osteogenesis induced by bone morphogenetic protein 9 (BMP9) in mesenchymal stem cell and to dissect the possible mechanism underlaying this effect. Methods This research employed histochemical staining to test the alkaline phosphatase (ALP) activities when treated the C3H10T1/2 cells with BMP9 and/or ATRA for 5 or 7 days. Then, evaluated the expression level of osteopontin by Western blot on D9 and D11, so did the mineralization with Alizarin Red Staining on D14 and D20. Finally, analyzed the activation of BMP-Smad signaling with BMPR-Smad binding site luciferase reporter assay and Western blot to determine the phosphorylation level of Smad1/5/8. Results The activities of ALP are increased by BMP9 and/or ATRA, but the ALP activities of BMP9 combine with ATR