Hypertrophy is a critical process for chondrocyte differentiation and maturation during endochondral ossification, which is responsible for the formation of long bone and postnatal longitudinal growth. Increasing evidence suggests that melatonin, an indole hormone, plays a pivotal role in chondrogenesis. However, little is known about the effects of melatonin on the terminal differentiation of chondrocytes. Mesenchymal stem cell (MSC)-derived chondrocytes generated by a high-density micromass culture system were induced to undergo hypertrophic differentiation. Melatonin-mediated hypertrophic differentiation was examined by reverse transcription polymerase chain reaction analysis (RT-PCR) analysis, histological staining and immunohistochemistry. Activation of the Wnt signaling pathway was evaluated by PCR array, RT-PCR, western blotting and immunofluorescence. XAV-939, a Wnt signaling pathway antagonist, was further used to determine whether the effect of melatonin on chondrocyte hypertrophic differentiation was mediated occurred by activation of Wnt signaling pathway. Histological staining showed melatonin increased chondrocyte cell volume and the expression of type X collagen but decreased the expression of type II collagen compared with the control group. RT-PCR showed that melatonin significantly up-regulated the gene expressions of biomarkers of hypertrophic chondrocytes, including type X collagen, alkaline phosphatase, runt-related transcription factor 2, Indian hedgehog and parathyroid hormone-related protein receptor, and melatonin down-regulated the mRNA expression of hallmarks of chondrocytes, including parathyroid hormone-related protein. PCR array showed that the effect of melatonin on chondrocyte hypertrophic differentiation was accompanied by the up-regulation of multiple target genes of the canonical Wnt signaling pathway, and this effect was blocked by XAV-939. The current findings demonstrate that melatonin enhances the hypertrophic differentiation of MSC-derived chondrocytes through the Wnt signaling pathway. Our findings add evidence to the role of melatonin in promoting bone development and highlight the positive effects of melatonin on terminal differentiation of chondrocytes.

中文翻译：

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褪黑激素通过激活 Wnt/β-catenin 信号通路促进间充质干细胞衍生的软骨细胞的肥大分化

肥大是软骨内骨化过程中软骨细胞分化和成熟的关键过程，负责长骨的形成和出生后的纵向生长。越来越多的证据表明褪黑激素，一种吲哚激素，在软骨形成中起着关键作用。然而，关于褪黑激素对软骨细胞终末分化的影响知之甚少。由高密度微团培养系统产生的间充质干细胞 (MSC) 衍生的软骨细胞被诱导进行肥大分化。通过逆转录聚合酶链反应分析 (RT-PCR) 分析、组织学染色和免疫组织化学检查褪黑激素介导的肥大分化。Wnt 信号通路的激活通过 PCR 阵列、RT-PCR、蛋白质印迹和免疫荧光。进一步使用Wnt信号通路拮抗剂XAV-939来确定褪黑激素对软骨细胞肥大分化的影响是否是由Wnt信号通路的激活介导的。组织学染色显示，与对照组相比，褪黑激素增加了软骨细胞体积和 X 型胶原蛋白的表达，但降低了 II 型胶原蛋白的表达。RT-PCR显示褪黑激素显着​​上调肥大软骨细胞生物标志物的基因表达，包括X型胶原、碱性磷酸酶、矮小相关转录因子2、印度刺猬和甲状旁腺激素相关蛋白受体，褪黑激素下调软骨细胞标志物的 mRNA 表达，包括甲状旁腺激素相关蛋白。PCR阵列显示，褪黑激素对软骨细胞肥大分化的影响伴随着经典Wnt信号通路多个靶基因的上调，而这种作用被XAV-939阻断。目前的研究结果表明，褪黑激素通过 Wnt 信号通路增强了 MSC 来源的软骨细胞的肥大分化。我们的研究结果为褪黑激素在促进骨骼发育中的作用提供了证据，并强调了褪黑激素对软骨细胞终末分化的积极影响。目前的研究结果表明，褪黑激素通过 Wnt 信号通路增强了 MSC 来源的软骨细胞的肥大分化。我们的研究结果为褪黑激素在促进骨骼发育中的作用提供了证据，并强调了褪黑激素对软骨细胞终末分化的积极影响。目前的研究结果表明，褪黑激素通过 Wnt 信号通路增强了 MSC 来源的软骨细胞的肥大分化。我们的研究结果为褪黑激素在促进骨骼发育中的作用提供了证据，并强调了褪黑激素对软骨细胞终末分化的积极影响。