Department of Orthopedics, The Fifth People's Hospital of Shanghai, Fudan University, China.
J Musculoskelet Neuronal Interact. 2022 Mar 1;22(1):113-122.
In bone tissue engineering, the use of osteoblastic seed cells has been widely adopted to mediate the osteogenic differentiation so as to prompt bone regeneration and repair. It is hypothesized that Dok5 can regulate the proliferation and differentiation of osteoblasts. In this study, the role of Dok5 in osteoblast proliferation and differentiation was investigated.
A lentiviral vector to silence Dok5 was transferred to C3H10, 293T and C2C12 cells. CCK-8 assay was used to detect the cell proliferation. Cells were stained by ALP and AR-S staining. Western blot and RT-PCR were used to detect the expression levels of related factors.
Dok5 expression level was gradually up-regulated during the osteoblast differentiation. Dok5 silencing down-regulated the expression levels of osteogenic biosignatures OPN, OCN, and Runx2 and suppressed the osteogenesis. Additionally, the osteoblast proliferation and canonical Wnt/β-catenin signaling were suppressed upon Dok5 knockdown, β-catenin expression level was significantly down-regulated in the knockdown group, while the expression levels of GSK3-β and Axin, negative regulators in the Wnt signaling pathway, were up-regulated. Furthermore, overexpression of Dok5 promoted the proliferation and osteogenesis and activated the canonical Wnt/β-catenin signaling pathway.
Dok5 may regulate the osteogenic proliferation and differentiation via the canonical Wnt/β-catenin signaling pathway.
在骨组织工程中，广泛采用成骨细胞种子细胞来介导成骨分化，以促进骨再生和修复。据推测，Dok5 可以调节成骨细胞的增殖和分化。本研究旨在探讨 Dok5 在成骨细胞增殖和分化中的作用。
通过慢病毒载体转染沉默 Dok5 的质粒至 C3H10、293T 和 C2C12 细胞，采用 CCK-8 法检测细胞增殖，通过碱性磷酸酶（ALP）和茜素红 S（ARS）染色检测细胞矿化，Western blot 和 RT-PCR 检测相关因子的表达水平。
在成骨细胞分化过程中，Dok5 的表达水平逐渐上调。沉默 Dok5 表达可下调成骨生物标志物 OPN、OCN 和 Runx2 的表达水平，抑制成骨作用。此外，Dok5 敲低抑制成骨细胞增殖和经典 Wnt/β-catenin 信号通路，β-catenin 表达水平在敲低组中显著下调，而 Wnt 信号通路的负调控因子 GSK3-β 和 Axin 的表达水平上调。此外，过表达 Dok5 可促进细胞增殖和成骨作用，并激活经典 Wnt/β-catenin 信号通路。
Dok5 可能通过经典 Wnt/β-catenin 信号通路调节成骨细胞的增殖和分化。
