BMP9通过Erk5信号通路调控间充质干细胞成骨分化

发布时间：2018-10-12 18:39

【摘要】：目的：初步分析丝裂原活化蛋白激酶ERK5在骨形成蛋白9诱导小鼠间充质干细胞C3H10T1/2、小鼠成骨细胞株MC3T3、小鼠成肌细胞株C2C12、小鼠胚胎成纤维细胞MEF成骨分化过程中的作用。方法：利用BMP9重组腺病毒感染C3H10T1/2、MC3T3、C2C12、MEF细胞，western blot检测ERK5激酶总蛋白表达情况和磷酸化情况。ERK5的特异性抑制剂S1531抑制ERK5活性后，分析ALP活性变化，利用茜素红S染色检测钙盐沉积，Real Time PCR检测Runx2和Smad7的mRNA表达水平，RT-PCR检测Id-1，Id-2，Id-3，CTGFmRNA表达水平，western blot检测Runx2，Smad1/5/8，，OPN，OCN蛋白表达水平，荧光素酶活性检测Smad1/5/8活性。结果： BMP9不影响ERK5激酶的蛋白表达水平，但却可以促进ERK5激酶的磷酸化；ERK5抑制剂S1531可剂量依赖性地抑制由BMP9诱导的C3H10T1/2、MC3T3、C2C12、MEF细胞的碱性磷酸酶（alkalinephosphatase, ALP）活性,S1531还可剂量依赖性地抑制BMP9诱导的C3H10T1/2、C2C12、MEF细胞的钙盐沉积； BMP9的靶基因Id-1，Id-2，Id-3，CTGF和Smad7的mRNA水平可以被ERK5特异性抑制剂S1531抑制；BMP9-Smad经典通路中的Smad1/5/8磷酸化水平和荧光素酶活性也被S1531所抑制；成骨相关重要转录因子Runx2的mRNA和蛋白水平也被S1531抑制；成骨相关OPN，OCN的蛋白水平也被S1531抑制。结论：ERK5信号通路在BMP9诱导的上述四种细胞向成骨细胞分化过程中起了作用。
[Abstract]:Aim: to investigate the role of mitogen-activated protein kinase (ERK5) in the osteogenic differentiation of mouse mesenchymal stem cells C3H10T1 / 2, mouse osteoblast strain MC3T3, mouse myoblast line C2C12 and mouse embryonic fibroblast cell line MEF induced by bone morphogenetic protein 9 (BMP9). Methods: the total protein expression and phosphorylation of ERK5 kinase were detected by, western blot in C3H10T1 / 2 MC3T3C2C12MEF cells infected with BMP9 recombinant adenovirus. S1531, a specific inhibitor of ERK5, inhibited the activity of ERK5 and analyzed the changes of ALP activity. Alizarin red S staining was used to detect the mRNA expression of Runx2 and Smad7 by calcium salt deposition, Real Time PCR, Id-1,Id-2,Id-3,CTGFmRNA expression level by RT-PCR, Runx2,Smad1/5/8,OPN,OCN protein expression by, western blot and Smad1/5/8 activity by luciferase activity. Results: BMP9 did not affect the protein expression of ERK5 kinase, but promoted the phosphorylation of ERK5 kinase. ERK5 inhibitor S1531 inhibited the alkaline phosphatase (alkalinephosphatase, ALP) activity of C3H10T1 / 2MC3T3C12MEF cells in a dose-dependent manner. S1531 also inhibited the calcium deposition of C3H10T1 / 2C12MEF cells induced by BMP9 in a dose-dependent manner. The mRNA levels of Id-1,Id-2,Id-3,CTGF and Smad7 were inhibited by ERK5 specific inhibitor S1531, and the phosphorylation of Smad1/5/8 and luciferase activity in BMP9-Smad pathway were also inhibited by S1531. The mRNA and protein levels of osteoblast-associated transcription factor Runx2 were also inhibited by S1531, and the protein level of osteoblast-associated OPN,OCN was also inhibited by S1531. Conclusion: ERK5 signaling pathway plays an important role in the differentiation of the four cells into osteoblasts 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】：2012
【分类号】：R329

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