本文選題：ST2 + caveolin-1　； 參考：《南昌大學(xué)》2012年碩士論文

【摘要】：目的：觀察P-catenin與caveolin-1相互作用在骨髓基質(zhì)干細(xì)胞ST2定向誘導(dǎo)成骨樣細(xì)胞分化成熟過程中的作用及其機制。 方法與結(jié)果：本研究首先采用western blotting法觀察ST2分化為成骨細(xì)胞過程不同時間點(第1,2,3,4,5,6天)caveolin-1和β-catenin蛋白的表達(dá)變化。并在誘導(dǎo)的第6天進(jìn)行成骨細(xì)胞cAKP染色鑒定。結(jié)果顯示ST2定向分化為成骨細(xì)胞,在分化過程中caveolin-1的蛋白表達(dá)無顯著差異,β-catenin蛋白的表達(dá)逐漸增加。采用western blotting法觀察ST2誘導(dǎo)的成骨細(xì)胞不同時間點(第3,6,9,12,15天)caveolin-1和β-catenin蛋白的表達(dá)變化,再用RT-PCR法觀察caveolin-1、β-catenin各時間點mRNA的含量變化。結(jié)果顯示caveolin-1蛋白在第1-9天表達(dá)較高,第9天開始逐漸減少；β-catenin蛋白的表達(dá)3-12天逐漸增加,第15天顯著減少。caveolin-1mRNA表達(dá)與蛋白表達(dá)呈現(xiàn)一致變化,β-catenin mRNA表達(dá)在第6天時明顯上調(diào),第9天時開始下調(diào),之后表達(dá)無顯著變化。在成骨細(xì)胞增殖期加入有效的RNA干擾質(zhì)粒pGenesil-1.1-cav-1下調(diào)caveolin-1表達(dá)后,β-catenin的蛋白表達(dá)減少；采用不同濃度的糖原合酶激酶3p(GSK-3p)抑制劑LiCl(5,10mM)抑制GSK-3β的活性。在成骨細(xì)胞分化期、礦化期加入LiCl caveolin-1蛋白表達(dá)無顯著變化,但LiCl組β-catenin蛋白表達(dá)高于對照組,并有濃度依賴性。此外,采用pNPP法測定成骨細(xì)胞分化第6,9,12,15天各處理組細(xì)胞裂解液中的堿性磷酸酶(ALP)含量發(fā)現(xiàn),與對照組相比高濃度LiCl組堿性磷酸酶活性有所上調(diào),其它各組與對照組相似。用VON KOSSA染色分別觀察成骨細(xì)胞分化第15,18天礦化結(jié)節(jié)情況卻發(fā)現(xiàn),不同濃度LiCl組礦化結(jié)節(jié)數(shù)量明顯低于對照組,高濃度LiC1組礦化結(jié)節(jié)數(shù)明顯少于低濃度組。 結(jié)論：β-catenin可以通過Wnt/β-catenin信號通路調(diào)節(jié)ST2誘導(dǎo)分化的成骨細(xì)胞的分化,對成骨細(xì)胞分化起促進(jìn)作用；caveolin-1可以通過Wnt/β-catenin途徑調(diào)節(jié)ST2誘導(dǎo)分化的成骨細(xì)胞的礦化過程�？赡苁峭ㄟ^下調(diào)caveolin-1蛋白即而使β-catenin的儲備量下降,以致消減礦化期Wnt/p-catenin信號,最終促進(jìn)成骨細(xì)胞礦化,caveolin-1和β-catenin對成骨細(xì)胞礦化起抑制作用。
[Abstract]:Objective: To observe the interaction between P-catenin and caveolin-1 on the differentiation and maturation of osteoblast like cells induced by ST2 in bone marrow stromal cells.
Methods and results: in this study, Western blotting was used to observe the changes in the expression of caveolin-1 and beta -catenin in the process of ST2 differentiation into osteoblasts at different time points (1,2,3,4,5,6 days). The osteoblasts were identified by cAKP staining at sixth days of induction. The results showed that ST2 was differentiated into osteoblast and caveolin during the differentiation process. There was no significant difference in the expression of -1 protein, and the expression of beta -catenin protein increased gradually. The expression of caveolin-1 and beta -catenin protein in ST2 induced osteoblasts at different time points (3,6,9,12,15 days) was observed by Western blotting. The content of mRNA in caveolin-1 and beta -catenin at each time point was observed by the RT-PCR method. The expression of -1 protein was higher on the 1-9 day and gradually decreased at the beginning of ninth days; the expression of beta -catenin protein increased gradually in 3-12 days. The expression of.Caveolin-1mRNA and protein expression showed a consistent change in fifteenth days. The expression of beta -catenin mRNA was obviously up-regulated at sixth days, and there was no significant change in expression after ninth days. When the effective RNA interference plasmid pGenesil-1.1-cav-1 was added, the expression of caveolin-1 was reduced, the protein expression of beta -catenin decreased; the activity of GSK-3 beta was inhibited by LiCl (5,10mM), a glycogen synthase kinase 3P (GSK-3p) inhibitor, LiCl (5,10mM) with different concentrations. There was no significant change in the expression of LiCl caveolin-1 protein at the stage of osteoblast differentiation and the addition of LiCl caveolin-1 protein in the mineralization period. In addition, the content of alkaline phosphatase (ALP) in the cell lysates of each treatment group after osteoblast differentiation on day 6,9,12,15 by pNPP method was found to be higher than the control group. The activity of alkaline phosphatase in the LiCl group of the high concentration LiCl group was up up, and the other groups were similar to the control group. VON KOSSA was used. The mineralization nodules on the 15,18 day of osteoblast differentiation were observed, but the number of mineralized nodules at different concentrations in LiCl group was significantly lower than that in the control group, and the number of mineralized nodules in the high concentration LiC1 group was significantly less than that in the low concentration group.
Conclusion: beta -catenin can regulate the differentiation of osteoblasts induced by ST2 through the Wnt/ beta -catenin signaling pathway and promote the differentiation of osteoblasts. Caveolin-1 can regulate the mineralization process of osteoblasts induced by ST2 through the Wnt/ beta -catenin pathway. It may be to store the beta -catenin by reducing the caveolin-1 protein. The amount of preparation decreased, so that the Wnt/p-catenin signal in mineralization stage was reduced, and the mineralization of osteoblasts was finally promoted. Caveolin-1 and beta -catenin inhibited the mineralization of osteoblasts.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329.2

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