摘要
目的研究西地那非对体外培养大鼠骨髓间充质干细胞(Rat bone marrow stromal stem cells,rBMSCs)增殖与成骨性分化的影响。方法取大鼠股骨及胫骨全骨髓,贴壁筛选法培养rBMSCs,每3天换液1次,9d后传代培养。采用终浓度分别为1×10-4、1×10-5、1×10-6、1×10-7mol·L-1的西地那非进行药物干预,MTT法检测细胞增殖率,同时检测成骨性诱导培养后9d的碱性磷酸酶(ALP)活性,筛选出最佳浓度。以最佳药物浓度干预rBMSCs的成骨性分化,比较西地那非组和不加药的对照组之间的钙化结节数量及阳性克隆数(colonies stained positive for alkaline phosphatase,CFU-FALP);提取Total RNA,Real-Time PCR检测Runx-2与Osterix mRNA的表达情况;Western blot法检测Ⅰ型胶原蛋白的分泌量。结果西地那非剂量依赖性抑制细胞的增殖,但可强烈促进成骨性分化,最佳药物浓度为1×10-5mol·L-1。该浓度下的西地那非可明显增加钙化结节和CFU-FALP数量,促进与成骨相关的转录因子Runx-2与Osterix mRNA的表达,同时也可增强Ⅰ型胶原蛋白的分泌。结论浓度为1×10-5mol·L-1的西地那非可明显促进rBMSCs的成骨性分化,提示西地那非在具备其他药理作用的同时,也有较强的抗骨质疏松活性,具备开发为抗骨质疏松药物的潜力。
Aim To investigate the effects of sildenafil on the proliferation and osteogenic differentiation of rat bone marrow stromal cells（rBMSCs）.Methods The rat bone marrow sample was obtained,and rBMSCs were cultured using adherence screening methods.The culture medium was replaced every three days.Nine days later,serial subcultivation proceeded.Sildenafil spplemented into the culture at 1×10-4,1×10-5,1×10-6,1×10-7mol·L-1 respectively.MTT method was adopted in proliferation analysis.The activity of ALP was examined after induced 9 days.Search the best concentration and supplemented into the medium,then the osteogenic differentiation markers including mineralized bone nodules,CFU-FALP were compared between the sildenafil-supplemented group and the control.Total RNA was isolated and the gene expression of Runx-2 and Osterix was investgated by RT-Real Time PCR.The total protein was also isolated and the secre-tion of collagenⅠ was examined by Western blot.Results The proliferation of rBMSCs was refrained by sildenafil dose-dependently.But it evidently led was to osteogenic process.1×10-5mol·L-1 was the best concentration.Sildenafil improved the secretion of collagen Ⅰ,CFU-FALP amounts and mineralized nodules significantly.Besides,it also could enhance the mRNA level of Runx-2 and Osterix.Conclusions The sildenafil with final concentration of 1×10-5mol·L-1 enhances the osteogenic differentiation of rBMSCs significantly,suggesting that sildenafil not only has other pharmacologic actions,but also has the activity of antiosteoporosis.It has the potential to be developed into a new drug of anti-osteoporosis.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2010年第11期1489-1494,共6页
Chinese Pharmacological Bulletin
基金
甘肃省科技重大专项计划资助项目(No092nkDA025)
