背景:干细胞分化为疾病治疗开辟了新的途径,组织修复,以及再生医学研究中的药物开发,具有巨大的应用前景。本研究旨在探讨槲皮素对间充质干细胞(MSCs)分化为成纤维细胞的作用机制。
方法:在本研究中,细胞分化实验和流式细胞术检测SD大鼠骨髓间充质干细胞的成功分离。5、10和20μM的槲皮素用作低,中等,和高剂量干预MSCs。使用CCK-8细胞计数试剂盒检测槲皮素处理后24、48和72小时韧带成纤维细胞的细胞活力变化。通过流式细胞术测定细胞增殖能力。RT-qPCR检测TGF-β1、IGF-1、COL-Ⅰ、COL-Ⅲ,FN(纤连蛋白),和不同实验组的TNMD(Tenomodulin)。通过分子对接实验探讨槲皮素对TGF-β1和IGF-1蛋白的结合作用。
结果:流式细胞术验证了MSCs的成功分离,CD29和CD73高表达,CD90和CD45低表达。实验结果表明,中、低剂量槲皮素能增强细胞增殖,而高剂量对细胞没有显著影响。通过流式细胞术检测细胞增殖产生与CCK-8相似的结果。Transwell实验表明,中低剂量槲皮素可以增加细胞迁移能力。此外,RT-qPCR检测显示槲皮素可增加TGF-β1和IGF-1的mRNA表达,促进COL-Ⅰ的表达,COL-Ⅲ,FN,和韧带成纤维细胞中的TNMD基因。分子对接结果显示槲皮素能与TGF-β1和IGF-1牢固结合。
结论:总体而言,本研究揭示了MSCs的形态特征和鉴定,以及槲皮素对韧带成纤维细胞行为的调控机制。槲皮素通过调节TGF-β1和IGF-1的表达影响韧带成纤维细胞的增殖和基因表达,可能为骨骼系统的生物医学研究提供新的视角。
BACKGROUND: Stem cell differentiation has opened up new avenues for disease treatment, tissue repair, and drug development in the
study of regenerative medicine, and has huge application prospects. This
study aimed to explore the mechanism of quercetin on the differentiation of mesenchymal stem cells (MSCs) into fibroblasts.
METHODS: In this
study, cell differentiation experiments and flow cytometry were used to detect the successful isolation of bone marrow MSCs from SD rats. Quercetin at 5, 10, and 20 μM was used as low, medium, and high doses to intervene in MSCs. The cell viability changes of ligament fibroblasts at 24, 48, and 72 hours after quercetin treatment were detected using a CCK-8 cell counting kit. Cell proliferative capacity was determined by flow cytometry. RT-qPCR measured the relative expression levels of TGF-β1, IGF-1, COL-Ⅰ, COL-Ⅲ, FN (fibronectin), and TNMD (Tenomodulin) in different experimental groups. Molecular docking experiments were conducted to explore the binding effect of quercetin on TGF-β1 and IGF-1 proteins.
RESULTS: Flow cytometry verified the successful isolation of MSCs, which had high expression of CD29 and CD73, while lower expression of CD90 and CD45. Experimental results show that low and medium doses of quercetin can enhance cell proliferation, while high doses have no significant effect on cells. Detection of cell proliferation through flow cytometry yielded similar results to CCK-8. Transwell experiments have shown that low and medium doses of quercetin can increase cell migration ability. In addition, RT-qPCR detection showed that quercetin can increase the mRNA expression of TGF-β1 and IGF-1, and promote the expression of COL-Ⅰ, COL-Ⅲ, FN, and TNMD genes in ligament fibroblasts. Molecular docking results showed that quercetin can bind firmly to TGF-β1 and IGF-1.
CONCLUSIONS: Overall, this
study revealed the morphological characteristics and identification of MSCs, as well as the regulatory mechanism of quercetin on the behavior of ligament fibroblasts. Quercetin affects the proliferation and gene expression of ligament fibroblasts by regulating the expression of TGF-β1 and IGF-1, which may provide a new perspective for biomedical research on the skeletal system.