活化α7乙酰胆碱受体促进LPS刺激的人牙髓干细胞牙/骨向分化
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国家自然科学基金(82270955);江苏省科教能力提升工程—江苏省研究型医院建设单位(YJXYYJSDW4);江苏省医学创新中心(CXZX202227)


Activation of alpha 7 nicotinic acetylcholine receptors promotes LPS⁃stimulated odontogenic/ osteogenic differentiation of human dental pulp stem cells
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    摘要:

    目的:探讨活化α7乙酰胆碱受体(alpha 7 nicotinic acetylcholine receptor,α7-nAChR)联合钙离子(calcium ion,Ca2+ ) 对LPS刺激的人牙髓干细胞(dental pulp stem cell,DPSC)牙/骨向分化的影响。方法:分离培养DPSC,流式细胞术对DPSC进行表面标志物表达鉴定。CCK-8检测α7-nAChR 激动剂PNU-282987和Ca2+ 对DPSC增殖的影响。通过碱性磷酸酶(alkaline phos- phatase,ALP)活性和染色筛选PNU-282987促进DPSC表达ALP活性的最佳浓度。用大肠杆菌脂多糖(lipopolysaccharide,LPS) 模拟炎性微环境刺激DPSC。采用免疫印迹分析(Western blot,WB)、实时定量聚合酶链反应(quantitative real-time polymerase chain reaction,RT-qPCR)和茜素红染色等方法检测牙/骨向分化的相关蛋白:Ⅰ型胶原(type Ⅰ collagen,COL-I)、牙本质涎磷蛋白(dentin sialoprotein,DSPP)、骨钙素(osteopontin,OPN)、ALP、核心转录因子-2(runt-related transcription factor 2,RUNX2)、成骨细胞特异性转录因子(osterix,OSX),相关基因(COL-I、DSPP、OPN、ALP、RUNX2、OSX)和矿化基质表达情况。Fura-2AM 用于检测细胞内Ca2+ 流动情况。结果:CCK-8 实验显示,PNU-282987 浓度低于 10 μmol/L 时对细胞增殖无抑制作用,且此浓度处理 LPS 刺激的 DPSC 后 ALP 活性增加最明显;Ca2+ 浓度低于 2 mmol/L 对细胞增殖无抑制作用;Western blot 和 RT-qPCR 实验显示,PNU-282987及Ca2+ 处理后的LPS刺激的DPSC牙/骨向分化相关蛋白(COL-I、DSPP、OPN、ALP、RUNX2、OSX)和相关基因(COL-I、DSPP、OPN、ALP、RUNX2、OSX)的表达及矿化基质形成均明显上调,二者联合后上调最显著(P < 0.001)。Fura-2 AM 钙离子探针结果显示DPSC细胞内Ca2+ 浓度增加。结论:10 μmol/L PNU-282987联合2 mmol/L Ca2+ 可以促进LPS刺激的DPSC 的牙/骨向分化能力。

    Abstract:

    Objective:To investigate the effect of activation of alpha 7 nicotinic acetylcholine receptors(α7-nAChRs)combined with calcium ion(Ca2+ )on the odontogenic/osteogenic differentiation of lipopolysaccharide(LPS)-stimulated human dental pulp stem cells (DPSCs). Methods:DPSCs were isolated and cultured,and surface marker expression of DPSCs was identified by flow cytometry. The effect of α7-nAChRs agonist PNU-282987 and Ca2+ on DPSCs proliferation were detected by CCK-8. The optimal concentration of PNU- 282987 to promote alkaline phosphatase(ALP)activity in DPSCs was determined through ALP activity and staining. E.coli lipoplysaccharide(LPS)was used to simulate inflammatory microenvironment stimulation of DPSCs. The expression of proteins:type Ⅰ collagen(COL-I),dentin sialoprotein(DSPP),osteopontin(OPN),ALP,runt-related transcription factor2(RUNX2),osterix(OSX), as well as the gene expression(COL-I,DSPP,OPN,ALP,RUNX2,OSX)and mineralized matrix related to odontogenic/osteogenic differentiation was examined by Western blot,quantitative real-time polymerase chain reaction(RT-qPCR)and Alizarin red staining.Fura-2 AM was used to detect intracellular Ca2+ flux. Results:The CCK-8 assay showed that PNU-282987 at a concentration of less than 10 μmol/L had no inhibitory effect on cell proliferation,and this concentration significantly increased ALP activity in LPS-stimulated DPSCs. Ca2+ at a concentration of less than 2 mmol/L had no inhibitory effect on cell proliferation;Western blot and RT-qPCR experiments showed that the expression of osteogenic/osteogenic related proteins(COL-I,DSPP,OPN,ALP,RUNX2,OSX), genes(COL-I,DSPP,OPN,ALP,RUNX2,OSX),and mineralized matrix formation in LPS-stimulated DPSCs were significantly upregulated after treated with PNU-282987 and Ca2+ ,with the most significant upregulation observed when the two were combined(P < 0.001). Fura-2 AM Ca2+ probe results revealed an increase in intracellular Ca2+ in DPSCs. Conclusion:10 μmol/L PNU-282987 combined with 2 mmol/L Ca2+ can promote the odontogenic/osteogenic differentiation of LPS-stimulated DPSCs.

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李梦圆,王宇萌,徐青清,关卓,卞成玥,江飞,张光东.活化α7乙酰胆碱受体促进LPS刺激的人牙髓干细胞牙/骨向分化[J].南京医科大学学报(自然科学版),2024,(2):145-153

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  • 收稿日期:2023-07-19
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  • 在线发布日期: 2024-02-05
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