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    黄芪甲苷Ⅳ对人牙周膜干细胞增殖和成骨分化影响

    Effect of the astragaloside Ⅳ on proliferation and osteogenic differentiation of human periodontal stem cells

      摘要
    • 摘要:
      目的 探讨黄芪甲苷Ⅳ(AS-Ⅳ)对人牙周膜干细胞(hPDLSCs)增殖和成骨分化的影响及作用机制。
      方法 刮取人的牙齿根中1/3的牙周膜，采用组织块法对hPDLSCs进行培养；流式细胞术鉴定hPDLSCs表面CD105、CD90、CD44、CD45和CD34表达；使用碱性磷酸酶(ALP)染色、茜素红(ARS)染色以及油红O染色，对细胞的多向分化能力(成骨和成脂分化)进行鉴定；hPDLSCs用不同浓度AS-Ⅳ(0、20、50、100、150 μmol/L)处理，通过CCK-8法检测hPDLSCs增殖活性；各组hPDLSCs(0、20、50、100、150 μmol/L AS-Ⅳ)共培养7、14 d，采用ALP染色和ALP活性检测及ARS染色检测不同浓度AS-Ⅳ对hPDLSCs成骨分化的影响；实验分为对照组和实验组(150 μmol/L AS-Ⅳ)，均用成骨诱导液培养14 d，通过qRT-PCR法检测ALP、Runt相关转录因子2(RUNX2)和骨钙素(OCN)的mRNA表达水平，Western blotting检测ALP、RUNX2、OCN和β-catenin蛋白表达水平。
      结果 hPDLSCs具备间充质干细胞特征，细胞表面标志物CD105、CD44和CD90均表现为阳性，而CD34、CD45显示为阴性；hPDLSCs可成骨分化和成脂分化，具有多向分化潜能；CKK-8实验显示，AS-Ⅳ促进hPDLSCs增殖(P < 0.05)；ALP染色和活性检测及ARS染色结果显示，AS-Ⅳ能促进人hPDLSCs的成骨分化；qRT-PCR和Western blotting结果显示，与对照组相比，AS-Ⅳ(150 μmol/L)组ALP、RUNX2、OCN mRNA和蛋白相对表达量均升高(P < 0.05~P < 0.01)，β-catenin蛋白表达降低(P < 0.05)。
      结论 AS-Ⅳ能促进hPDLSCs成骨分化，其机制可能为通过抑制Wnt/β-catenin信号通路实现。

       

      Abstract:
      Objective To investigate the effects of astragaloside Ⅳ(AS-Ⅳ) on proliferation and osteogenic differentiation of human periodontal stem cells(hPDLSCs) and its mechanism.
      Methods After removing one-third of the human periodontal ligament tissue, the hPDLSCs were cultured using the tissue block method.The expression of CD105, CD90, CD44, CD45, and CD34 on the surface of hPDLSCs were identifiied using flow cytometry.Alkaline phosphatase(ALP), alizarin red(ARS) and oil red O staining were used to identify the multidirection differentiation(osteogenic and lipogenic differentiation) of cells.The hPDLSCs were treated with different concentrations of AS-Ⅳ(0, 20, 50, 100, 150 μmol/L), and the proliferative activity of hPDLSCs was detected by CCK-8 method.The hPDLSCs were co-cultured with 0, 20, 50, 100, 150 μmol/L AS-Ⅳ for 7 and 14 days, and the effects of different AS-Ⅳ concentrations on osteogenic differentiation of hPDLSCs were detected by ALP staining, ALP activity detection and ARS staining.The experiment was divided into the control group and experimental group(150 μmol/L AS-Ⅳ), two groups were cultured with osteogenic induction solution for 14 days.The mRNA expression levels of ALP, Runt-related transcription factor 2(RUNX2) and osteocalcin(OCN) were detected by qRT-PCR.The expression levels of ALP, RUNX2, OCN and β-catenin proteins were detected by Western blotting.
      Results The hPDLSCs possessed mesenchymal stem cell properties, the surface antigens CD105, CD44, and CD90 were positive, but the CD34 and CD45 were negative.These cells had the capacity to differentiate in multiple directions, and can differentiate into osteogenesis and adipogenesis.AS-Ⅳ could promote the osteogenic differentiation of hPDLSCs.The results of qRT-PCR and Western blotting showed that compared with the control group, the relative expression levels of ALP, RUNX2 and OCN mRNA and protein in AS-Ⅳ(150 μmol/L) group increased(P < 0.05 to P < 0.01), and the expression level of β-catenin protein decreased(P < 0.05).
      Conclusions AS-Ⅳ can promote the osteogenic differentiation of hPDLSCs, which may be achieved by inhibiting Wnt/β-catenin signaling pathway.

       

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