RUNX1对BMP9诱导间充质干细胞成骨分化的影响
发布时间：2018-12-15 08:53
【摘要】：目的:探究RUNX1对BMP9诱导间充质干细胞成骨分化的影响。方法:用Ad-BMP9腺病毒感染间充质干细胞,RT-PCR和Western Blot分别在mRNA和蛋白水平检测RUNX1的内源性表达。构建过表达RUNX1(Ad-RUNX1)和干扰RUNX1(Ad-siRUNX1)的重组腺病毒,并在mRNA和蛋白水平验证Ad-RUNX1和Ad-siRUNX1的效果。用Ad-RUNX1或Ad-siRUNX1分别与BMP9条件培养基共处理间充质干细胞,碱性磷酸酶染色和活性测定检测成骨早期分化指标ALP,茜素红S染色检测成骨晚期分化指标钙盐沉积,RT-q PCR检测成骨相关转录因子RUNX2、DLX5、OSX、CollaI和成骨标志基因OCN在mRNA水平的变化,Western Blot检测RUNX2、DLX5和OCN在蛋白水的变化。最后用Ad-RUNX1、Ad-siRUNX1和Ad-BMP9处理MEFs细胞,检测经典的BMPs/Smad信号通路中Smad1/5/8和MAPK信号通路中p38和ERK1/2在总蛋白和磷酸化水平的变化。结果:Ad-BMP9可使RUNX1在mRNA和蛋白水平表达上调。构建的重组腺病毒Ad-RUNX1和Ad-siRUNX1能有效在mRNA和蛋白水平过表达或干扰间充质干细胞中RUNX1的表达。Ad-RUNX1和Ad-siRUNX1处理BMP9诱导的间充质干细胞后,Ad-RUNX1增强了BMP9诱导的ALP活性、钙盐沉积、成骨相关转录因子RUNX2、DLX5、OSX、CollaI和成骨标志基因OCN在mRNA水平的表达,RUNX2、DLX5和OCN蛋白水平的改变与mRNA水平一致。Ad-siRUNX1减弱了BMP9诱导的ALP活性、钙盐沉积、成骨相关转录因子RUNX2、DLX5、OSX、CollaI和成骨标志基因OCN在mRNA水平的表达,RUNX2、DLX5和OCN蛋白水平的改变与mRNA水平一致。用Ad-RUNX1、Ad-siRUNX1和Ad-BMP9处理MEFs细胞后,过表达RUNX1增强BMP9诱导的Smad1/5/8的磷酸化,干扰RUNX1抑制BMP9诱导的Smad1/5/8的磷酸化,总的Smad1/5/8不受RUNX1影响。RUNX1不影响BMP9诱导的p38和ERK1/2的磷酸化水平。结论:RUNX1可促进BMP9诱导的间充质干细胞成骨分化。
[Abstract]:Objective: to investigate the effect of RUNX1 on the osteogenic differentiation of mesenchymal stem cells induced by BMP9. Methods: Ad-BMP9 adenovirus infected mesenchymal stem cells, RT-PCR and Western Blot were used to detect the endogenous expression of RUNX1 at the mRNA and protein levels, respectively. Recombinant adenovirus expressing RUNX1 (Ad-RUNX1) and interfering RUNX1 (Ad-siRUNX1) was constructed, and the effects of Ad-RUNX1 and Ad-siRUNX1 were tested at mRNA and protein levels. Mesenchymal stem cells were co-treated with Ad-RUNX1 or Ad-siRUNX1 with BMP9 conditioned medium. Alkaline phosphatase staining and activity measurement were used to detect the early differentiation index of osteogenesis, ALP, alizarin red S staining, and calcium salt deposition in late osteogenic differentiation index. RT-q PCR detection of osteoblast-associated transcription factor RUNX2,DLX5,OSX,CollaI and osteoblastic marker gene OCN at the mRNA level the changes of RUNX2,DLX5 and OCN in protein water were detected by, Western Blot. Finally, MEFs cells were treated with Ad-RUNX1,Ad-siRUNX1 and Ad-BMP9 to detect the changes of total protein and phosphorylation levels of p38 and ERK1/2 in Smad1/5/8 and MAPK signaling pathways. Results: Ad-BMP9 could up-regulate the expression of RUNX1 at the level of mRNA and protein. The recombinant adenovirus Ad-RUNX1 and Ad-siRUNX1 could effectively overexpress or interfere with the expression of RUNX1 in mesenchymal stem cells at the mRNA and protein levels. Ad-RUNX1 and Ad-siRUNX1 treated BMP9 induced mesenchymal stem cells. Ad-RUNX1 enhanced BMP9 induced ALP activity, calcium deposition, osteoblast-associated transcription factor RUNX2,DLX5,OSX,CollaI and osteoblastic marker OCN expression at mRNA level, RUNX2, Changes in DLX5 and OCN protein levels were consistent with mRNA levels. Ad-siRUNX1 reduced BMP9 induced ALP activity, calcium deposition, osteoblast-associated transcription factor RUNX2,DLX5,OSX,CollaI and osteoblastic marker OCN expression at mRNA level, RUNX2, The changes of DLX5 and OCN protein levels were consistent with mRNA levels. After MEFs cells were treated with Ad-RUNX1,Ad-siRUNX1 and Ad-BMP9, overexpression of RUNX1 enhanced Smad1/5/8 phosphorylation induced by BMP9 and interfered with RUNX1 inhibited Smad1/5/8 phosphorylation induced by BMP9. Total Smad1/5/8 was not affected by RUNX1. RUNX1 did not affect the phosphorylation levels of p38 and ERK1/2 induced by BMP9. Conclusion: RUNX1 can promote the differentiation of mesenchymal stem cells induced by BMP9.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R329.2
本文编号：2380368
[Abstract]:Objective: to investigate the effect of RUNX1 on the osteogenic differentiation of mesenchymal stem cells induced by BMP9. Methods: Ad-BMP9 adenovirus infected mesenchymal stem cells, RT-PCR and Western Blot were used to detect the endogenous expression of RUNX1 at the mRNA and protein levels, respectively. Recombinant adenovirus expressing RUNX1 (Ad-RUNX1) and interfering RUNX1 (Ad-siRUNX1) was constructed, and the effects of Ad-RUNX1 and Ad-siRUNX1 were tested at mRNA and protein levels. Mesenchymal stem cells were co-treated with Ad-RUNX1 or Ad-siRUNX1 with BMP9 conditioned medium. Alkaline phosphatase staining and activity measurement were used to detect the early differentiation index of osteogenesis, ALP, alizarin red S staining, and calcium salt deposition in late osteogenic differentiation index. RT-q PCR detection of osteoblast-associated transcription factor RUNX2,DLX5,OSX,CollaI and osteoblastic marker gene OCN at the mRNA level the changes of RUNX2,DLX5 and OCN in protein water were detected by, Western Blot. Finally, MEFs cells were treated with Ad-RUNX1,Ad-siRUNX1 and Ad-BMP9 to detect the changes of total protein and phosphorylation levels of p38 and ERK1/2 in Smad1/5/8 and MAPK signaling pathways. Results: Ad-BMP9 could up-regulate the expression of RUNX1 at the level of mRNA and protein. The recombinant adenovirus Ad-RUNX1 and Ad-siRUNX1 could effectively overexpress or interfere with the expression of RUNX1 in mesenchymal stem cells at the mRNA and protein levels. Ad-RUNX1 and Ad-siRUNX1 treated BMP9 induced mesenchymal stem cells. Ad-RUNX1 enhanced BMP9 induced ALP activity, calcium deposition, osteoblast-associated transcription factor RUNX2,DLX5,OSX,CollaI and osteoblastic marker OCN expression at mRNA level, RUNX2, Changes in DLX5 and OCN protein levels were consistent with mRNA levels. Ad-siRUNX1 reduced BMP9 induced ALP activity, calcium deposition, osteoblast-associated transcription factor RUNX2,DLX5,OSX,CollaI and osteoblastic marker OCN expression at mRNA level, RUNX2, The changes of DLX5 and OCN protein levels were consistent with mRNA levels. After MEFs cells were treated with Ad-RUNX1,Ad-siRUNX1 and Ad-BMP9, overexpression of RUNX1 enhanced Smad1/5/8 phosphorylation induced by BMP9 and interfered with RUNX1 inhibited Smad1/5/8 phosphorylation induced by BMP9. Total Smad1/5/8 was not affected by RUNX1. RUNX1 did not affect the phosphorylation levels of p38 and ERK1/2 induced by BMP9. Conclusion: RUNX1 can promote the differentiation of mesenchymal stem cells induced by BMP9.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R329.2
【参考文献】
相关期刊论文 前1条
1 Mengrui Wu;Guiqian Chen;Yi-Ping Li;;TGF-β and BMP signaling in osteoblast,skeletal development,and bone formation,homeostasis and disease[J];Bone Research;2016年01期
，本文编号：2380368
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