Effect of oxymatrine on expression of stem markers and osteogenic differentiation of periodontal ligament stem cells

doi:10.12307/2025.056

Abstract

Abstract: BACKGROUND: Human periodontal ligament stem cells are potential functional cells for periodontal tissue engineering. However, long-term in vitro culture may lead to reduced stemness and replicative senescence of periodontal ligament stem cells, which may impair the therapeutic effect of human periodontal ligament stem cells.
OBJECTIVE: To investigate the effect of oxymatrine on the stemness maintenance and osteogenic differentiation of periodontal ligament stem cells in vitro, and to explore the potential mechanism.
METHODS: Periodontal ligament stem cells were isolated from human periodontal ligament tissues by tissue explant enzyme digestion and cultured. The surface markers of mesenchymal cells were identified by flow cytometry. Periodontal ligament stem cells were incubated with 0, 2.5, 5, and 10 μg/mL oxymatrine. The effect of oxymatrine on the proliferation activity of periodontal ligament stem cells was detected by CCK8 assay. The appropriate drug concentration for subsequent experiments was screened. Western blot assay was used to detect the expression of stem cell non-specific proteins SOX2 and OCT4 in periodontal ligament stem cells. qRT-PCR and western blot assay were used to detect the expression levels of related osteogenic genes and proteins in periodontal ligament stem cells.
RESULTS AND CONCLUSION: (1) The results of CCK8 assay showed that 2.5 μg/mL oxymatrine significantly enhanced the proliferative activity of periodontal stem cells, and the subsequent experiment selected 2.5 μg/mL oxymatrine to intervene. (2) Compared with the blank control group, the protein expression level of SOX2, a stem marker of periodontal ligament stem cells in the oxymatrine group did not change significantly (P > 0.05), and the expression of OCT4 was significantly up-regulated (P < 0.05). (3) Compared with the osteogenic induction group, the osteogenic genes ALP, RUNX2 mRNA expression and their osteogenic associated protein ALP protein expression of periodontal ligament stem cells were significantly down-regulated in the oxymatrine + osteogenic induction group (P < 0.05). (4) The oxymatrine up-regulated the expression of stemness markers of periodontal ligament stem cells and inhibited the bone differentiation of periodontal ligament stem cells, and the results of high-throughput sequencing showed that it may be associated with WNT2, WNT16, COMP, and BMP6.

Key words: periodontal ligament stem cell, matrine alkaloids, osteogenesis, cell proliferation, oxymatrine, stemness maintenance, high-throughput sequencing

CLC Number:

Luo Jing, Yong Min, Chen Qi, Yang Changyi, Zhao Tian, Ma Jing, Mei Donglan, Hu Jinpeng, Yang Zhaojun, Wang Yuran, Liu Bo. Effect of oxymatrine on expression of stem markers and osteogenic differentiation of periodontal ligament stem cells[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(19): 3992-3999.

Figures/Tables 2

2.1 牙周膜干细胞形态、表面标志物表达及骨向分化能力鉴定人牙周组织块在T25培养瓶内培养，5-7 d后在倒置显微镜下可见组织块周围长出纺锤形细胞，细胞排列稀疏不均匀，生长较慢，见图1A。第3代牙周膜干细胞在倒置显微镜下呈长梭形，生长较快，排列紧密，见图1B；第3代牙周膜干细胞成骨诱导21 d后进行茜素红染色，镜下可见培养板底部有橙红色结节形成，说明分离出来的细胞具有成骨分化能力，见图1C。流式细胞仪检测牙周膜干细胞阳性表达间充质干细胞表面标志物CD73、CD90、CD105；阴性表达白细胞共同抗原CD45，体现了间充质干细胞的干性特点，见图1D。 2.2 氧化苦参碱对牙周膜干细胞增殖能力的影响 CCK8实验检测0，2.5，5，10 μg/mL氧化苦参碱孵育1，3，5，7 d后细胞增殖活性变化，用于评估氧化苦参碱对牙周膜干细胞增殖能力的影响。如图2所示，随着时间的延长，各组细胞数量呈增加趋势。第1天时，10 μg/mL氧化苦参碱组细胞数量低于2.5 μg/mL氧化苦参碱组(P < 0.05)；第3天时组间没有明显差异；第5天时，10 μg/mL氧化苦参碱组细胞数量低于2.5 μg/mL氧化苦参碱组(P < 0.01)；第7天时，4组细胞生长趋势减缓，2.5 μg/mL氧化苦参碱组细胞数量高于0 μg/mL氧化苦参碱组(P < 0.05)。综上，初步选择低质量浓度2.5 μg/mL氧化苦参碱进行后续实验。 2.3 氧化苦参碱作用下牙周膜干细胞中干细胞标志物的表达培养7 d后，采用Western blot检测空白对照组和氧化苦参碱组牙周膜干细胞中干细胞标志物SOX2和OCT4 的表达。由图3所示，氧化苦参碱组SOX2蛋白表达较空白对照组无明显变化(P > 0.05)；与空白对照组相比，氧化苦参碱组OCT4蛋白表达明显上升，差异有显著性意义(P < 0.05)。 2.4 氧化苦参碱作用下牙周膜干细胞成骨诱导后成骨相关基因和蛋白表达水平与空白对照组相比，成骨诱导组早期成骨基因ALP表达明显上调，表明牙周膜干细胞成骨分化诱导成功，由于培养时间短，故成骨诱导组中RUNX2基因表达差异不明显；与成骨诱导组相比，氧化苦参碱+成骨诱导组牙周膜干细胞成骨相关基因RUNX2(P < 0.05)、ALP(P < 0.000 1)表达明显降低，以上实验结果说明氧化苦参碱可以抑制牙周膜干细胞的成骨向分化，见图4A，B。同期，Western blot实验结果表明，成骨相关蛋白ALP的表达趋势与ALP成骨基因表达的趋势一样，即：与空白对照组相比，成骨诱导组早期成骨相关蛋白ALP表达水平明显上调(P < 0.000 1)；与成骨诱导组相比，氧化苦参碱+成骨诱导组牙周膜干细胞成骨相关蛋白ALP的表达水平显著下降(P < 0.000 1)，见图4C，D。"

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