磷酸二氢钾通过核因子-κB 通路促进人牙周膜干细胞的增殖和分化。
Potassium dihydrogen phosphate promotes the proliferation and differentiation of human periodontal ligament stem cells via nuclear factor kappa B pathway.
机构信息
Endodontic Department, Changzhou Stomatological Hospital, 61 Beizhi Street, Changzhou, Jiangsu 213000, China; Key Laboratory of Oral Diseases of Jiangsu Province and Stomatological Institute of Nanjing Medical University, 140 Hanzhong Road, Nanjing, Jiangsu 210029, China.
Key Laboratory of Oral Diseases of Jiangsu Province and Stomatological Institute of Nanjing Medical University, 140 Hanzhong Road, Nanjing, Jiangsu 210029, China; Endodontic Department, School of Stomatology, Nanjing Medical University, 136 Hanzhong Road, Nanjing, Jiangsu 210029, China.
出版信息
Exp Cell Res. 2019 Nov 1;384(1):111593. doi: 10.1016/j.yexcr.2019.111593. Epub 2019 Sep 2.
INTRODUCTION
Periodontal ligament stem cells (PDLSCs) are vital for the regeneration of periodontal tissues. Potassium dihydrogen phosphate (KHPO) has recently been applied as a component of the mineralization inducing medium (MM), which can be used to induce osteogenic differentiation of dental stem cells. However, whether KHPO has effects on PDLSCs has not been studied.
MATERIALS AND METHODS
PDLSCs were isolated by magnetic activated cell sorting and cultured. Alkaline phosphatase (ALP) activity and ALP protein expression of PDLSCs treated with different concentrations of KHPO were examined to make sure the optimal concentration of KHPO for the following experiments. The effects of KHPO on the proliferation and differentiation of PDLSCs were investigated by flow cytometry, cell counting kit-8 assay, alizarin red staining, real-time RT-PCR, and Western blot. The involvement of nuclear factor kappa B (NF-κB) pathway in KHPO-treated PDLSCs was analyzed by Western blot and alizarin red staining.
RESULTS
ALP activity assay and ALP protein expression examination revealed that 1.8 mmol/L KHPO was the optimal concentration for the induction of hPDLSCs by KHPO. The proliferation and mineralization capacity of PDLSCs treated with KHPO were enhanced as compared with the control group. PDLSCs treated with KHPO showed an improved proliferation capacity in logarithmic growth phase at day 7. As PDLSCs were treated with KHPO, the expression of odonto/osteogenic markers (OCN/OCN, DSP/DSPP, OSX/OSX, RUNX2/RUNX2, and ALP/ALP) in cells were up-regulated at day 3 or 7. Moreover, the expression of IκBα in cytoplasm was down-regulated, along with an increased expression of p-P65 in cytoplasm and an up-regulated expression of P65 in nucleus. When treated with BMS345541 (the specific NF-κB inhibitor), the odonto/osteogenic differentiation of KHPO-treated PDLSCs was significantly attenuated.
CONCLUSION
KHPO can improve the proliferation and odonto/osteogenic differentiation capacity of PDLSCs via NF-κB pathway, and thus represents a potential target involved in the regeneration of periodontium for clinical treatments.
简介
牙周膜干细胞（PDLSCs）是牙周组织再生的关键。最近，磷酸二氢钾（KHPO）已被用作矿化诱导培养基（MM）的成分之一，可用于诱导牙源性干细胞的成骨分化。然而，KHPO 是否对 PDLSCs 有影响尚未被研究。
材料与方法
通过磁激活细胞分选分离和培养 PDLSCs。通过碱性磷酸酶（ALP）活性和 ALP 蛋白表达检测不同浓度 KHPO 处理后的 PDLSCs，以确定后续实验中 KHPO 的最佳浓度。通过流式细胞术、细胞计数试剂盒-8 测定、茜素红染色、实时 RT-PCR 和 Western blot 研究 KHPO 对 PDLSCs 增殖和分化的影响。通过 Western blot 和茜素红染色分析 KHPO 处理后的 PDLSCs 中核因子 kappa B（NF-κB）通路的参与情况。
结果
ALP 活性测定和 ALP 蛋白表达检测显示，1.8mmol/L KHPO 是 KHPO 诱导 hPDLSCs 的最佳浓度。与对照组相比，用 KHPO 处理后的 PDLSCs 的增殖和矿化能力增强。KHPO 处理后的 PDLSCs 在第 7 天的对数生长期显示出改善的增殖能力。随着 PDLSCs 用 KHPO 处理，细胞中牙/骨形成标志物（OCN/OCN、DSP/DSPP、OSX/OSX、RUNX2/RUNX2 和 ALP/ALP）的表达在第 3 天或第 7 天上调。此外，细胞质中 IκBα 的表达下调，同时细胞质中 p-P65 的表达增加，核中 P65 的表达上调。用 BMS345541（特异性 NF-κB 抑制剂）处理时，KHPO 处理后的 PDLSCs 的牙/骨形成分化明显减弱。
结论
KHPO 可通过 NF-κB 通路改善 PDLSCs 的增殖和成牙/骨分化能力，因此代表了参与牙周组织再生的潜在临床治疗靶点。
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