Li Siyu, Jia Xiang, Cui Yongping, Liu Kainan, Xu Tianjie, Fan Jiaxin, Zhang Hui, Li Yueyuan, Wang Qian
School of Basic Medical Sciences, North China University of Science and Technology, Tangshan, 063210, Hebei, China.
School of Basic Medical Sciences, Xingtai Medical College, Xingtai, 054000, Hebei, China.
Cell Tissue Bank. 2025 Jun 26;26(3):30. doi: 10.1007/s10561-025-10180-5.
The purpose of this study was to investigate the role of Calcium-Sensing Receptor in the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and to provide a new target for cartilage defect repair. BMSCs were cultured in vitro, and cultured in the complete culture medium with gradient concentration of calcium sensitive receptor inhibitor and activator, and the optimum dose was selected by CCK-8 experiment. The experiment was divided into four groups. After 7, 14 and 21 days of intervention, the intracellular calcium concentration was detected by laser confocal microscope, the differentiation of cartilage was detected by toluidine blue staining, and the expression of cartilage marker proteins (Col- II, Agg and Sox9) was detected by immunocytochemical staining and Western Blot. The CCK-8 assay results showed that the optimal concentrations of Gd and NPS were 300 μM and 10 μM, respectively. After 7, 14, and 21 days of culture, intracellular calcium fluorescence decreased, with notably higher cartilage differentiation in the NPS inhibitor group. Col-II, Agg and Sox9 chondrocyte marker proteins increased with culture time in all groups, with significantly higher levels in the inhibitor group compared to others, followed by the cartilage induction solution group, and then the activator group. Inhibition of calcium sensitive receptors can promote chondrogenic differentiation of rat BMSCs by regulating Sox9, affecting Col- II and Agg.
本研究旨在探讨钙敏感受体在骨髓间充质干细胞（BMSCs）成软骨分化中的作用，为软骨缺损修复提供新靶点。体外培养BMSCs，在含有梯度浓度钙敏感受体抑制剂和激活剂的完全培养基中培养，通过CCK-8实验筛选最佳剂量。实验分为四组。干预7、14和21天后，用激光共聚焦显微镜检测细胞内钙浓度，用甲苯胺蓝染色检测软骨分化情况，用免疫细胞化学染色和蛋白质免疫印迹法检测软骨标记蛋白（Col-II、Agg和Sox9）的表达。CCK-8检测结果显示，Gd和NPS的最佳浓度分别为300 μM和10 μM。培养7、14和21天后，细胞内钙荧光降低，NPS抑制剂组软骨分化明显更高。所有组中Col-II、Agg和Sox9软骨细胞标记蛋白均随培养时间增加，抑制剂组水平显著高于其他组，其次是软骨诱导液组，然后是激活剂组。抑制钙敏感受体可通过调节Sox9、影响Col-II和Agg促进大鼠BMSCs的成软骨分化。
