Zunyi Municipal Key Laboratory of Medicinal Biotechnology, Center for Translational Medicine, Affiliated Hospital of Zunyi Medical University, 149 Dalian Road, Zunyi 563003, PR China.
Department of Laboratory Medicine, Affiliated Hospital of Zunyi Medical University, 149 Dalian Road, Zunyi 563003, PR China.
Life Sci. 2019 Sep 1;232:116669. doi: 10.1016/j.lfs.2019.116669. Epub 2019 Jul 18.
This study investigated the effects of hyaluronic acid (HA), a commonly used osteogenic medium referred to as DAG, and the combined administration of HA and DAG (CG) on the osteogenic differentiation of human amniotic mesenchymal stem cells (hAMSCs), and the underlying mechanism.
The phenotype of hAMSCs was detected by flow cytometry and immunocytochemical staining. Alkaline phosphatase (ALP) and calcium deposition assays were employed for evaluating the osteogenic differentiation of hAMSCs. The expression of osteogenesis-related genes and proteins was determined by quantitative reverse transcription PCR (qRT-PCR) and Western blotting, respectively. Meanwhile, the molecular mechanism of osteogenic differentiation of hAMSCs was detected by PCR array and qRT-PCR.
The results showed that treatment with CG could significantly stimulate hAMSC ALP activity and calcium deposition compared to treatment with DAG, while HA had little effect. The expression of osteogenesis-related molecules and stemness-related molecules was up-regulated at the mRNA and protein levels in all three groups, and this up-regulation was most significant in the CG group. In addition, treatment with CG significantly increased the gene expressions involved in regulation of the TGF-β/Smad signalling pathway compared to treatment with DAG. Furthermore, the pro-osteogenic differentiation effects as well as the up-regulated expression of genes observed in the CG treatment group were significantly inhibited when the cells were pre-treated with SB431542, an inhibitor of the TGF-β/Smad pathway.
These results suggest that HA in combination with DAG could significantly enhance the osteogenic differentiation of hAMSCs, potentially via the TGF-β/Smad signalling pathway.
本研究旨在探讨透明质酸（HA），一种常用的成骨培养基，即 DAG，以及 HA 和 DAG 联合应用（CG）对人羊膜间充质干细胞（hAMSCs）成骨分化的影响及其潜在机制。
通过流式细胞术和免疫细胞化学染色检测 hAMSCs 的表型。碱性磷酸酶（ALP）和钙沉积测定用于评估 hAMSCs 的成骨分化。通过定量逆转录 PCR（qRT-PCR）和 Western blot 分别测定成骨相关基因和蛋白的表达。同时，通过 PCR 阵列和 qRT-PCR 检测 hAMSCs 成骨分化的分子机制。
结果表明，与 DAG 处理相比，CG 处理可显著刺激 hAMSC 的 ALP 活性和钙沉积，而 HA 作用不大。三组中，成骨相关分子和干性相关分子的表达均在 mRNA 和蛋白水平上调，CG 组上调最为显著。此外，与 DAG 处理相比，CG 处理显著增加了 TGF-β/Smad 信号通路调节相关基因的表达。此外，当用 TGF-β/Smad 通路抑制剂 SB431542 预处理细胞时，CG 处理组的促成骨分化作用以及观察到的基因表达上调均受到显著抑制。
这些结果表明，HA 与 DAG 联合应用可显著增强 hAMSCs 的成骨分化，可能通过 TGF-β/Smad 信号通路。
