淫羊藿苷促进大鼠骨髓基质细胞成骨性分化过程中对p-AKT及eNOS、iNOS表达量的影响  

The expression of p-AKT,eNOS and iNOS in the process of ICA promotes osteogenic differentiation of BMSCs

作　　者：郭晓宇[1] 葛宝丰[1] 陈克明[1] 甄平[1] 周建[1] 马小妮[1] 

机构地区：[1]兰州军区兰州总医院骨科研究所,甘肃兰州730050

出　　处：《中国骨质疏松杂志》2013年第9期897-901,906,共6页Chinese Journal of Osteoporosis

基　　金：甘肃省科技重大专项资助项目(09ZNKDA025);国家自然科学基金(81270963)

摘　　要：目的研究淫羊藿苷(Icariin,ICA)促进体外培养大鼠骨髓基质细胞(Bone marrow stromal cells,BMSCs)的成骨性分化机理。方法贴壁分离法培养大鼠骨髓基质细胞并用流式检测细胞纯度。骨髓基质细胞传代至第2代时分别用含有成骨性诱导剂的1×10-5mol/L ICA和LY294002处理24 h后用Real-time RT-PCR检测ALP、eNOS和iNOS的mRNA表达水平,Western blotting检测ALP、p-AKT、eNOS和iNOS的蛋白表达量。结果原代骨髓基质细胞体外培养传代至第2代时纯度可达95%以上。基因和蛋白质检测均显示ICA可极显著的促进碱性磷酸酶(ALP)的表达(P<0.01),PI3K的特异性抑制剂LY294002会抑制ICA对ALP的提高,ICA+LY294002组与CON组的表达差别不明显。p-AKT的蛋白表达变化与ALP相似。ICA可以提高eNOS表达(P<0.01),加抑制剂能使其降低(P<0.01),而iNOS的ICA组与ICA+LY294002组无明显差别。结论 ICA促进BMSCs成骨性分化过程涉及到PI3K/AKT通路,且AKT位于eNOS的上游;eNOS和iNOS共同作用,但iNOS起主要作用。Objective To investigate the effect mechanism of Icariin （ICA） on osteogenic differentiation of bone marrow stromal cells （ BMSCs）. Methods Rat BMSCs were isolated according to plastic adherence and identified by flow cytometry. The mRNA levels of alkaline phosphatase （ALP）, eNOS, and iNOS were detected using quantitative real-time RT-PCR. The protein levels of ALP, p- AKT, eNOS, and iNOS were detected using by Western blotting. Results More than 95% of the 2na passage rat BMSCs were immune positive, with less than 5% were immune negative. The result showed that 10-3 mol/L ICA could significantly promote the expression of ALP （ P 〈 0. 01 ）, while the expression in LY294002 group was lower than that in the ICA group （ P 〈 0.01 ）. The expression in ICA ＋ LY294002 group and control group had no significant difference. The protein levels of p-AKT were similar to ALP. The expression of eNOS in ICA group increased （P 〈 0.01 ） , while decreased in LY294002 group （P 〈 0. 01 ）. However, no significant difference of protein level was observed between ICA group and ICA ＋ LY294002 group. Conclusion ICA promotes osteogenie differentiation of BMSCs, and the PI3K/AKT signal pathway is involved in this process. AKT is located at the upstream of eNOS. eNOS and iNOS both can affect the process, while iNOS may play a major role.

关 键 词：大鼠骨髓基质细胞 淫羊藿苷 NOS AKT 

分 类 号：R681[医药卫生—骨科学]