caveolin-1及β-catenin相互作用参与骨髓基质细胞系ST2定向分化为成骨细胞及其机制研究

发布时间：2018-05-24 05:52

本文选题：ST2 + caveolin-1　；参考：《南昌大学》2012年硕士论文

【摘要】：目的：观察P-catenin与caveolin-1相互作用在骨髓基质干细胞ST2定向诱导成骨样细胞分化成熟过程中的作用及其机制。方法与结果：本研究首先采用western blotting法观察ST2分化为成骨细胞过程不同时间点(第1,2,3,4,5,6天)caveolin-1和β-catenin蛋白的表达变化。并在诱导的第6天进行成骨细胞cAKP染色鉴定。结果显示ST2定向分化为成骨细胞,在分化过程中caveolin-1的蛋白表达无显著差异,β-catenin蛋白的表达逐渐增加。采用western blotting法观察ST2诱导的成骨细胞不同时间点(第3,6,9,12,15天)caveolin-1和β-catenin蛋白的表达变化,再用RT-PCR法观察caveolin-1、β-catenin各时间点mRNA的含量变化。结果显示caveolin-1蛋白在第1-9天表达较高,第9天开始逐渐减少；β-catenin蛋白的表达3-12天逐渐增加,第15天显著减少。caveolin-1mRNA表达与蛋白表达呈现一致变化,β-catenin mRNA表达在第6天时明显上调,第9天时开始下调,之后表达无显著变化。在成骨细胞增殖期加入有效的RNA干扰质粒pGenesil-1.1-cav-1下调caveolin-1表达后,β-catenin的蛋白表达减少；采用不同浓度的糖原合酶激酶3p(GSK-3p)抑制剂LiCl(5,10mM)抑制GSK-3β的活性。在成骨细胞分化期、矿化期加入LiCl caveolin-1蛋白表达无显著变化,但LiCl组β-catenin蛋白表达高于对照组,并有浓度依赖性。此外,采用pNPP法测定成骨细胞分化第6,9,12,15天各处理组细胞裂解液中的碱性磷酸酶(ALP)含量发现,与对照组相比高浓度LiCl组碱性磷酸酶活性有所上调,其它各组与对照组相似。用VON KOSSA染色分别观察成骨细胞分化第15,18天矿化结节情况却发现,不同浓度LiCl组矿化结节数量明显低于对照组,高浓度LiC1组矿化结节数明显少于低浓度组。结论：β-catenin可以通过Wnt/β-catenin信号通路调节ST2诱导分化的成骨细胞的分化,对成骨细胞分化起促进作用；caveolin-1可以通过Wnt/β-catenin途径调节ST2诱导分化的成骨细胞的矿化过程。可能是通过下调caveolin-1蛋白即而使β-catenin的储备量下降,以致消减矿化期Wnt/p-catenin信号,最终促进成骨细胞矿化,caveolin-1和β-catenin对成骨细胞矿化起抑制作用。
[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.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329.2

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