Estrogen regulates osteogenic differentiation of human periodontal ligament stem cells via Wnt/beta-catenin signaling pathway

doi:10.3969/j.issn.2095-4344.0498

Abstract

Abstract:

BACKGROUND: Estrogen can promote the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs), but the molecular mechanism is unclear.
OBJECTIVE: To study the regulatory effect of estrogen on the osteogenic differentiation of hPDLSCs via Wnt/β-catenin signaling pathway.
METHODS: The hPDLSCs were isolated and purified by digestion method combined with limited dilution clone method. Three experimental groups were set as follows: osteogenic induction only (control group); 1×10^-⁷ mol/L estrogen with osteogenic induction (estrogen group); and

100 μg/L Wnt3a protein with osteogenic induction (Wnt3a group). Alkaline phosphatase activity was detected at 1, 3, 5, 7 days of osteogenic induction. Western blot was used to detect the expression of Wnt/β-catenin signaling pathway related proteins β-catenin, P-GSK-3β, GSK-3β, CyclinD1 and osteoblast-related proteins Runx2 and OCN after 7 days of osteogenic induction.
RESULTS AND CONCLUSION: The activity of ALP in all groups increased with time. The expression level of ALP in the estrogen group and Wnt3a group was higher than that in the control group at 1, 3, 5 and 7 days of induction (P < 0.05), while there was no significant difference between the former two groups (P > 0.05). The western blot results showed that the expression levels of β-catenin, P-GSK-3β, CyclinD1, Runx2 and OCN in the estrogen group and Wnt3a group were higher than those in the control group (P < 0.05), while the expression of GSK-3β was lower than that in the control group (P < 0.05). But there were no differences in the expression of Wnt/β-catenin signaling pathway related proteins and mid-late osteogenic markers between estrogen group and Wnt3a group (P > 0.05). To conclude, estrogen can enhance the osteogenic differentiation of hPDLSCs, and the underlying mechanism is likely to activate the Wnt/β-catenin signaling pathway in activated hPDLSCs exposed to estrogen.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Periodontal Ligament, Stem Cells, Estrogens, Cell Differentiation, Osteoblasts, Tissue Engineering

CLC Number:

R394.2

Mao Jie, Zhou Yi-fei, Wu Xiao-ling, Yu Jing-hong, Dang Hai-xia, Xu Xiao-mei. Estrogen regulates osteogenic differentiation of human periodontal ligament stem cells via Wnt/beta-catenin signaling pathway[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(13): 2087-2092.

Figures/Tables 2

2.1 hPDLSCs的分离培养和纯化采用消化组织块法培养原代细胞，5-10 d可见组织块周围有原代细胞爬出(图1A)，进一步通过有限稀释克隆法得到纯化的hPDLSCs。纯化后的hPDLSCs形态丰满呈长梭形，胞核聚集在中心，细胞呈放射状、漩涡状生长，2周左右可达80%汇合(图1B)。 2.2 hPDLSCs的鉴定 2.2.1 流式分析hPDLSCs表面抗原表达流式细胞仪检测结果显示第3代hPDLSCs阳性表达间质细胞表面标记物Stro-1(14.21%)、CD146(99.15%)、CD90(99.55%)，阴性表达造血细胞表面标记物CD45(0.15%)、CD31(0.49%)，证实所取得细胞为间充质来源(图2)。 2.2.2 免疫组化染色鉴定结果第3代hPDLSCs抗波形蛋白(vimentin)染色阳性，细胞胞浆呈均匀棕黄色(图3A)；抗角蛋白(Cytokeratin)染色为阴性(图3B)，证实所取得的细胞为间充质来源而非上皮来源。 2.2.3 细胞多向分化能力第3代hPDLSCs经过21 d成骨诱导后茜素红染色呈阳性，光镜下可见大量矿化结节的形成(图4A)；第3代hPDLSCs经过21 d成脂诱导后油红O染色呈阳性，光镜下可见脂滴生成(图4B)，证实hPDLSCs具有较强的多向分化潜能。 2.3 碱性磷酸酶活性各组hPDLSCs成骨诱导1，3，5，7 d检测碱性磷酸酶表达量。结果显示随时间推移，3组碱性磷酸酶的表达量均呈上升趋势。雌激素组和Wnt3a组各时间点碱性磷酸酶的表达量均明显高于对照组(P < 0.05)，而雌激素组和Wnt3a组间各时间点的表达量差异无显著性意义(P > 0.05，表1，图5)。 2.4 Western blot检测Wnt/β-catenin信号通路及成骨相关蛋白表达成骨诱导7 d后采用Western blot检测对照组与实验组Wnt/β-catenin信号通路相关蛋白β-catenin、GSK-3β、P-GSK-3β、CyclinD1及成骨相关蛋白Runx-2、OCN表达(图6)，采用Quantity one软件计算各目的蛋白灰度值与内参GAPDH灰度值比值，得到各目的蛋白相对表达量(表2，图7)，雌激素组和Wnt3a组相较于对照组各目的蛋白相对表达量差异均有显著性意义(P < 0.05)，而雌激素组和Wnt3a组间各目的蛋白相对表达量差异无显著性意义(P > 0.05)。"

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