【摘要】：目的：初步分析絲裂原活化蛋白激酶ERK5在骨形成蛋白9誘導(dǎo)小鼠間充質(zhì)干細(xì)胞C3H10T1/2、小鼠成骨細(xì)胞株MC3T3、小鼠成肌細(xì)胞株C2C12、小鼠胚胎成纖維細(xì)胞MEF成骨分化過(guò)程中的作用。方法：利用BMP9重組腺病毒感染C3H10T1/2、MC3T3、C2C12、MEF細(xì)胞，western blot檢測(cè)ERK5激酶總蛋白表達(dá)情況和磷酸化情況。ERK5的特異性抑制劑S1531抑制ERK5活性后，分析ALP活性變化，利用茜素紅S染色檢測(cè)鈣鹽沉積，Real Time PCR檢測(cè)Runx2和Smad7的mRNA表達(dá)水平，RT-PCR檢測(cè)Id-1，Id-2，Id-3，CTGFmRNA表達(dá)水平，western blot檢測(cè)Runx2，Smad1/5/8，，OPN，OCN蛋白表達(dá)水平，熒光素酶活性檢測(cè)Smad1/5/8活性。 結(jié)果： BMP9不影響ERK5激酶的蛋白表達(dá)水平，但卻可以促進(jìn)ERK5激酶的磷酸化；ERK5抑制劑S1531可劑量依賴性地抑制由BMP9誘導(dǎo)的C3H10T1/2、MC3T3、C2C12、MEF細(xì)胞的堿性磷酸酶（alkalinephosphatase, ALP）活性,S1531還可劑量依賴性地抑制BMP9誘導(dǎo)的C3H10T1/2、C2C12、MEF細(xì)胞的鈣鹽沉積； BMP9的靶基因Id-1，Id-2，Id-3，CTGF和Smad7的mRNA水平可以被ERK5特異性抑制劑S1531抑制；BMP9-Smad經(jīng)典通路中的Smad1/5/8磷酸化水平和熒光素酶活性也被S1531所抑制；成骨相關(guān)重要轉(zhuǎn)錄因子Runx2的mRNA和蛋白水平也被S1531抑制；成骨相關(guān)OPN，OCN的蛋白水平也被S1531抑制。 結(jié)論：ERK5信號(hào)通路在BMP9誘導(dǎo)的上述四種細(xì)胞向成骨細(xì)胞分化過(guò)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

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