The 2th Department of Orthopaedics, The First Hospital Of Qinhuangdao, Qinghuangdao, Heibei, China.
The 2th Department of Orthopaedics, The First Hospital Of Qinhuangdao, Qinghuangdao, Heibei, China.
Gene. 2024 Sep 25;923:148575. doi: 10.1016/j.gene.2024.148575. Epub 2024 May 17.
Steroid-induced osteonecrosis of the femoral head (SONFH) is a disease characterized by a collapsed femoral head caused by the overuse of glucocorticoids. Dysfunction of bone marrow mesenchymal stem cells (BMSCs) is an important pathological feature of SONFH. In this study, we investigated whether exosomes from SHEDs (stem cells from human exfoliated deciduous teeth) have a therapeutic effect on glucocorticoid-induced inhibition of proliferation and osteogenesis in BMSCs, and elucidated the underlying mechanisms involved.
Primary dental pulp cells were isolated and cultured from human deciduous tooth pulp, SHEDs were isolated and purified by the limiting dilution method and exosomes were isolated from the supernatants of SHEDs by ultracentrifugation. The cell surface markers CD31, CD34, CD45, CD73, CD90 and CD105 were detected by flow cytometry. A Cell-Counting-Kit-8 assay was used to detect cell activity. ALP and Alizarin Red staining were used to identify osteogenic differentiation ability, and exosomes were identified using transmission electron microscopy, NanoFCM and Western blotting. PKH67 fluorescence was used to track the uptake of exosomes by BMSCs. Transcriptome analysis combined with quantitative real-time PCR was used to explore the underlying mechanism involved.
Exosomes secreted by SHEDs can be endocytosed by BMSCs, and can partially reverse the inhibitory effects of glucocorticoids on the viability and osteogenic differentiation of BMSCs. Transcriptome sequencing analysis revealed that the differentially expressed mRNAs regulated by SHED-derived exosomes were enriched mainly in signaling pathways such as the apoptosis pathway, the PI3K-Akt signaling pathway, the Hippo signaling pathway and the p53 signaling pathway. qPCR showed that SHED-derived exosomes reversed the dexamethasone-induced upregulation of HGF and ITGB8 expression and the inhibition of EFNA1 expression, but further increased the dexamethasone-induced downregulation of IL7 expression. In conclusion, SHED-derived exosomes partially reversed the inhibitory effects of glucocorticoids on BMSC proliferation and osteogenesis by inhibiting the expression of HGF, ITGB8 and IL7, and upregulating the expression of EFNA1.
激素诱导性股骨头坏死（SONFH）是一种由糖皮质激素过度使用导致股骨头塌陷的疾病。骨髓间充质干细胞（BMSCs）功能障碍是 SONFH 的重要病理特征。在这项研究中，我们研究了来源于人乳牙牙髓的干细胞（SHEDs）的外泌体是否对糖皮质激素诱导的 BMSCs 增殖和成骨作用抑制具有治疗作用，并阐明了相关的潜在机制。
从人乳牙牙髓中分离和培养原代牙髓细胞，通过有限稀释法分离和纯化 SHEDs，并通过超速离心从 SHEDs 的上清液中分离外泌体。通过流式细胞术检测细胞表面标志物 CD31、CD34、CD45、CD73、CD90 和 CD105。使用细胞计数试剂盒-8 法检测细胞活性。碱性磷酸酶（ALP）和茜素红染色用于鉴定成骨分化能力，使用透射电子显微镜、纳米流式细胞术和 Western blot 鉴定外泌体。PKH67 荧光用于跟踪 BMSCs 对外泌体的摄取。通过转录组分析结合实时定量 PCR 探索潜在的相关机制。
SHEDs 分泌的外泌体可被 BMSCs 内吞，并可部分逆转糖皮质激素对 BMSCs 活力和成骨分化的抑制作用。转录组测序分析显示，SHED 衍生的外泌体调节的差异表达 mRNA 主要富集在凋亡通路、PI3K-Akt 信号通路、Hippo 信号通路和 p53 信号通路等信号通路中。qPCR 显示，SHED 衍生的外泌体逆转了地塞米松诱导的 HGF 和 ITGB8 表达上调以及 EFNA1 表达抑制，但进一步增加了地塞米松诱导的 IL7 表达下调。总之，SHED 衍生的外泌体通过抑制 HGF、ITGB8 和 IL7 的表达和上调 EFNA1 的表达，部分逆转了糖皮质激素对 BMSC 增殖和成骨作用的抑制。
