摘要
目的明确wnt信号通路对心脏瓣膜成肌纤维细胞向成骨细胞样表型转化的调控作用。方法以原代培养的猪主动脉瓣成肌纤维细胞作为研究对象,用与心脏瓣膜钙化有关的病理因素氧化型低密度脂蛋白(ox-LDL)处理细胞24h、48h、72h,在实验中加入wnt信号阻滞剂DDK-1与ox-LDL共同作用72h。采用免疫印迹法检测细胞的α-平滑肌蛋白(α-SMA)活性变化,骨形成蛋白2(BMP2)、碱性磷酸酶(ALP)和成骨细胞转录因子Cbfa-1的蛋白表达;利用免疫细胞化学法检测Wnt/β-catenin信号通路中的关键效应蛋白β-catenin蛋白质表达的变化,观察Wnt/β-catenin信号通路的变化以及细胞是否向成骨细胞样表型转化。结果BMP2、ALP和Cbfa-1蛋白在对照组没有或仅有极弱的表达;经ox-LDL处理细胞后,α-SMA、BMP2、ALP和Cbfa-1表达显著增加(P〈0.01),并呈现时间依赖性的表达上调(P%0.05);β-catenin蛋白的表达随着ox-LDL作用时间的增加而上调(P〈0.05),并由细胞质转移至胞核。加入Wnt通路阻滞剂DKK-1后,pcatenin蛋白及骨相关蛋白的表达显著下调(P〈O.05)。结论Wnt/β-catenin信号通路很可能在心脏瓣膜成肌纤维细胞向成骨细胞样表型转化的过程中起着关键性调控作用。
Objective To elucidate the role of the Wnt/β-catenin signaling pathway in regulating the phenotypic transformation of aortic valvular myofibroblasts to osteoblastqike cells. Methods Cultured primary valvular myofibroblastes isolated from porcine aortic valve leaflets were treated with oxidized low-density lipoprotein (ox-LDL) for different lengths of time: 24 h, 48 h and 72 h. The Wnt signaling pathway inhibitor Dickkopf-1 (DDK-1) was co-incubated with ox-LDL for 72 h. After cells harvest, the expression of myofibroblastic or osteoblast-like phenotype related markers, α-smooth muscle actin (α-SMA), bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP) and core- binding factora-1 (Cbα-1), was detected by Western blotting. The expression and sub-cellular localization of β-catenin was assessed by immunocytochemistry. Changes of the Wnt/β-catenin signaling pathway and the transformation of aortic valvular myofibroblast to osteoblast-like cells were monitored. Results BMP2, ALP and Cbfa-1 protein expression was not or barely detectable in the control group. However, after ox-LDL treatment, the expression of α-SMA, BMP2, ALP and Cbfα-1 increased significantly (each P〈0.01) in a time-dependent manner (each P〈0.05). Besides, ox-LDL was also able to up-regulate the protein expression of β--catenin in a time-dependent manner (P〈0. 05) and promoted its nuclear translocation. After DKK-1 treatment, the protein expression ofβ-catenin and osteogenesis-related markers was down-regulated (P〈0.05). Conclusions The Wnt/β-catenin signaling pathway may play a crucial role in regulating the transformation of aortic valvular myofibroblasts to an osteoblast-like phenotype.
出处
《中华老年医学杂志》
CAS
CSCD
北大核心
2016年第2期209-213,共5页
Chinese Journal of Geriatrics
基金
国家自然科学基金项目(81070190)
湖北省自然科学基金项目(2014CFB962)