黏着斑激酶诱导小鼠胚胎成纤维细胞成骨分化的作用及机制

Role and mechanism of focal adhesion kinase in inducing osteogenic differentiation of mouse embryonic fibroblasts cells

背景:黏着斑激酶被作为骨组织工程中“生物材料/支架”“种子细胞”“生长因子”3个要素的桥梁分子,研究其在相关种子细胞成骨分化过程中的作用及机制,对于骨组织工程的发展及应用尤为重要。目的:探讨黏着斑激酶在诱导永生化小鼠胚胎成纤维细胞成骨分化过程中的作用及机制。方法:在相同的诱导条件下诱导野生型及黏着斑激酶敲除后小鼠胚胎成纤维细胞成骨分化,在相同的诱导条件下诱导经磷脂酰肌醇3-激酶特异性磷酸化抑制剂和细胞外调节蛋白激酶磷酸化抑制剂处理的野生型及黏着斑激酶敲除后小鼠胚胎成纤维细胞成骨分化。在显微镜下观察不同诱导时期细胞形态改变,蛋白免疫印迹技术检测早期成骨相关蛋白表达水平及相应的磷脂酰肌醇3-激酶、丝氨酸/苏氨酸蛋白激酶、细胞外调节蛋白激酶磷酸化水平,Real-time PCR检测成骨相关基因转录水平,最后在成骨诱导3周后对诱导细胞进行茜素红钙结节染色。结果与结论:①相同的诱导条件下黏着斑激酶敲除后小鼠胚胎成纤维细胞的成骨效应较保留斑激酶的小鼠胚胎成纤维细胞组明显下降;②加入磷脂酰肌醇3-激酶特异性磷酸化抑制剂处理的野生型及黏着斑激酶敲除后,小鼠胚胎成纤维细胞与对照组相比成骨相关蛋白表达水平及成骨效应明显下降;③加入细胞外调节蛋白激酶磷酸化抑制剂处理的野生型及黏着斑激酶敲除后,小鼠胚胎成纤维细胞与对照组相比成骨相关蛋白表达水平及成骨效应明显下降;④提示静默黏着斑激酶表达可通过降低磷脂酰肌醇3-激酶、丝氨酸/苏氨酸蛋白激酶、细胞外调节蛋白激酶(1/2)磷酸化水平,降低成骨相关蛋白表达水平,进而抑制小鼠胚胎成纤维细胞成骨分化。

BACKGROUND:Focal adhesion kinase(FAK)is regarded as a bridge molecule of“biomaterial/scaffold,”“seed cell,”and“growth factor”in bone tissue engineering.Exploration on the role and mechanism of focal adhesion kinase in inducing osteogenic differentiation of related seed cells is particularly important for the development and application of bone tissue engineering.OBJECTIVE:To determine the role and mechanism of FAK in inducing osteogenic differentiation of immortalized mouse embryonic fibroblasts(iMEF).METHODS:Under the same induction conditions,the iMEF cells with(iMEFFAK+/+cells)or without FAK knockout(iMEF^FAK-/-cells),treated with or without PI3K/AKT phosphorylation inhibitor LY294002 or ERK1/2 phosphorylation inhibitor U0126,were induced to differentiate into osteoblasts.The morphological changes of iMEFs(iMEF^FAK+/+and iMEF^FAK-/-)at different induction periods were observed under a microscope.Runx2 protein levels and corresponding p-ERK1/2 and p-AKT levels were detected by western blot.RT-PCR technology was used to detect the transcription level of Runx2 gene.Finally,the induced iMEFs(iMEFFAK+/+and iMEF^FAK-/-)were stained with alizarin red staining for calcium nodules 3 weeks after osteogenesis induction.RESULTS AND CONCLUSION:The osteogenic effect of iMEF^FAK-/-cells was lower than that of iMEF^FAK+/+cells under the same induction conditions.Both the expression levels of Runx2 and the osteogenic effect of iMEF^FAK+/+cells and iMEF^FAK-/-cells treated with LY294002 decreased significantly compared with the control group.Both the expression levels of Runx2 and the osteogenic effect of iMEF^FAK+/+cells and iMEF^FAK-/-cells treated with U0126 decreased significantly compared with the control group.To conclude,silencing FAK expression can inhibit osteogenic differentiation of mouse embryonic fibroblasts by reducing the levels of PI3K/AKT,serine/threonine protein kinase,and ERK1/2 phosphorylation levels.