不同剂量TGF-β1在BMP9诱导间充质干细胞成骨分化中的作用

发布时间：2018-04-09 15:08

  本文选题：骨形成蛋白　切入点：转化成因子β　出处：《第三军医大学学报》2014年02期

【摘要】：目的分析联合应用不同剂量TGF-β1对BMP9诱导间充质干细胞成骨分化过程的影响,寻找最佳的促进BMP9成骨的TGF-β1剂量,为联合不同细胞因子促进组织工程骨再生寻找有效途径。方法应用鼠间充质干细胞株C3H10T1/2,以重组腺病毒法将其导入BMP9,再分别施加0、5、10、20 ng/mL TGF-β1蛋白处理,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(alkaline 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(collagenⅠ,COLⅠ)、骨桥素(osteopontin,OPN)、骨钙素(osteocalcin,OCN)的mRNA转录表达水平和蛋白表达水平改变;流式细胞仪检测细胞周期;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 d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/mL TGF-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/mL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。BMP9促进细胞增殖,TGF-β1对该效应起抑制作用,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。
[Abstract]:Objective to investigate the effects of different doses of TGF- 尾 1 on the osteogenic differentiation of mesenchymal stem cells (MSCs) induced by BMP9, to find the best dose of TGF- 尾 1 for promoting bone regeneration in tissue engineering with different cytokines.Methods the mouse mesenchymal stem cell line C3H10T1 / 2 was introduced into BMP9 by recombinant adenovirus method, and then treated with 0H101020 ng/mL TGF- 尾 1 protein respectively. The osteogenic differentiation model of mesenchymal stem cells induced by BMP9 combined with TGF- 尾 1 was established.The supernatant of TGF- 尾 1 protein with good activity was obtained by conditioned medium method, the expression and activity of alkaline phosphatase (ALP) were detected by modified SEAP chemiluminescence assay and histochemical staining, and calcium salt deposition was detected by alizarin red S staining.Real-time fluorescence quantitative PCR and Western blot were used to detect the changes of mRNA transcription and protein expression of osteoblast-associated gene type 鈪，

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