RUNX1對BMP9誘導(dǎo)間充質(zhì)干細胞成骨分化的影響
發(fā)布時間：2018-12-15 08:53
【摘要】：目的:探究RUNX1對BMP9誘導(dǎo)間充質(zhì)干細胞成骨分化的影響。方法:用Ad-BMP9腺病毒感染間充質(zhì)干細胞,RT-PCR和Western Blot分別在mRNA和蛋白水平檢測RUNX1的內(nèi)源性表達。構(gòu)建過表達RUNX1(Ad-RUNX1)和干擾RUNX1(Ad-siRUNX1)的重組腺病毒,并在mRNA和蛋白水平驗證Ad-RUNX1和Ad-siRUNX1的效果。用Ad-RUNX1或Ad-siRUNX1分別與BMP9條件培養(yǎng)基共處理間充質(zhì)干細胞,堿性磷酸酶染色和活性測定檢測成骨早期分化指標(biāo)ALP,茜素紅S染色檢測成骨晚期分化指標(biāo)鈣鹽沉積,RT-q PCR檢測成骨相關(guān)轉(zhuǎn)錄因子RUNX2、DLX5、OSX、CollaI和成骨標(biāo)志基因OCN在mRNA水平的變化,Western Blot檢測RUNX2、DLX5和OCN在蛋白水的變化。最后用Ad-RUNX1、Ad-siRUNX1和Ad-BMP9處理MEFs細胞,檢測經(jīng)典的BMPs/Smad信號通路中Smad1/5/8和MAPK信號通路中p38和ERK1/2在總蛋白和磷酸化水平的變化。結(jié)果:Ad-BMP9可使RUNX1在mRNA和蛋白水平表達上調(diào)。構(gòu)建的重組腺病毒Ad-RUNX1和Ad-siRUNX1能有效在mRNA和蛋白水平過表達或干擾間充質(zhì)干細胞中RUNX1的表達。Ad-RUNX1和Ad-siRUNX1處理BMP9誘導(dǎo)的間充質(zhì)干細胞后,Ad-RUNX1增強了BMP9誘導(dǎo)的ALP活性、鈣鹽沉積、成骨相關(guān)轉(zhuǎn)錄因子RUNX2、DLX5、OSX、CollaI和成骨標(biāo)志基因OCN在mRNA水平的表達,RUNX2、DLX5和OCN蛋白水平的改變與mRNA水平一致。Ad-siRUNX1減弱了BMP9誘導(dǎo)的ALP活性、鈣鹽沉積、成骨相關(guān)轉(zhuǎn)錄因子RUNX2、DLX5、OSX、CollaI和成骨標(biāo)志基因OCN在mRNA水平的表達,RUNX2、DLX5和OCN蛋白水平的改變與mRNA水平一致。用Ad-RUNX1、Ad-siRUNX1和Ad-BMP9處理MEFs細胞后,過表達RUNX1增強BMP9誘導(dǎo)的Smad1/5/8的磷酸化,干擾RUNX1抑制BMP9誘導(dǎo)的Smad1/5/8的磷酸化,總的Smad1/5/8不受RUNX1影響。RUNX1不影響B(tài)MP9誘導(dǎo)的p38和ERK1/2的磷酸化水平。結(jié)論:RUNX1可促進BMP9誘導(dǎo)的間充質(zhì)干細胞成骨分化。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R329.2
本文編號：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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R329.2
【參考文獻】
相關(guān)期刊論文 前1條
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
，本文編號：2380368
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