转化生长因子tgf-β1促进骨形成蛋白bmp-9诱导 - 第三军医大学学报.doc

不同剂量TGFβ1在BMP9诱导间充质干细胞成骨分化中的作用重庆医科大学附属第二医院骨科， [摘要目的联合应用不同剂量TGF-β1对BMP9诱导间充质干细胞成骨分化过程的影响。方法应用鼠间充质干细胞株C3H10T1/2，以重组腺病毒法将导入BMP9，再施加不同浓度TGF-β1处理，建立BMP9联合TGF-β1诱导间充质干细胞成骨分化模型。条件培养基法获取含活性良好的TGF-β1蛋白上清液；改良SEAP化学发光法和组织化学染色法检测碱性磷酸酶(lkaline phosphatase, ALP)表达及活性；茜素红S染色检测钙盐沉积；实时荧光定量PCR和Western blot法分别检测成骨相关基因Ⅰ型胶原(ollagenⅠ,COLⅠ)、骨桥素(steopotin, OPN)、骨钙素(steocalcin, OCN)的mRNA转录表达水平和蛋白表达水平改变；流式细胞术检测细胞周期，台盼蓝拒染法计数活细胞监测细胞增殖。结果5 ng/mL TGF-β1与BMP9联合处理较BMP9单独处理能更早更多地提高ALP活性和引起钙盐沉积，持续处理17后钙盐沉积结果不再显示明显差异，TGF-β1单独处理与对照组间无明显差异。随TGF-β1剂量增加，ALP活性和钙盐沉积效应受到抑制。5 ng/mTGF-β1与BMP9联合处理较BMP9单独处理使COLⅠ、OPN、OCN的mRNA转录水平和蛋白表达水平明显增高，COLⅠ增高变化发生较早和显著；TGF-β1应用剂量达到20 ng/m出现基因转录和蛋白表达受到抑制，但COLⅠ升高趋势仍能维持。TGF-β1对BMP9诱导成骨过程中的细胞增殖呈抑制效应，细胞周期转化表现G0/G1期阻滞。结论在BMP9诱导间充质干细胞成骨分化中，适量的TGF-β1联合应用具有促进作用；BMP9与TGF-β1在诱导成骨分化中可能存在协同作用关系。 关键词骨形成蛋白9；转化成因子(1；间充质干细胞；分化；成骨 [基金项目] 国家自然科学基金面上项目31000434) The affects of different concentrations of TGF-β1 on BMP9-induced osteogenesis in MSCs Li Ruidong1; Deng Zhongliang1*; Zhang Yamei2; Wang Yang1; Yin Liangjun1; Chen Liang1 （1Department of Orthopaedics, 2Department of anesthesiology, the Second affiliated Hospital, Chongqing Medical University, Chongqing, 400010, China） [Abstract] Objective The effectiveness of BMP9-induced osteogenic differentiation of MSCs combined with TGF(1 is investigated. Methods: Utilizing C3H10T1/2 cell line to construct the osteogenic cell model infected with recombinant adenovirus of BMP9 and stimulated with different concentrations of TGF(1 protein, which harvested by the conditioned medium method. The alkaline phosphatase (ALP) activity is assayed by the modified SEAP chemoluminescence assay and histochemical staining assay. Alizarin Red S staining is used for Matrix mineralization. Real-time PCR detects the mRNA transcription of collagen type1a2 (COL1a2), osteopotin (OPN) and osteocalcin (OCN). Western blotting is utilized for COL1A1, OPN and OCN. Viable cells count with trypan blue and flow cytometry are managed to find the correlation of cel