Ma Yu, Li Shu-Hui, Ding Xin-Xin, Wu Pei-Ling
Dept. of Stomatology, The 2nd Affiliated Hospital of Xinjiang Medical University, Urumqi 830063, China.
Hua Xi Kou Qiang Yi Xue Za Zhi. 2018 Apr 1;36(2):184-189. doi: 10.7518/hxkq.2018.02.013.
To evaluate the effects of tumor necrosis factor-α (TNF-α) on osteogenic differentiation and Notch signaling pathway of periodontal ligament stem cells (PDLSCs) and to investigate the regulatory role of Notch signaling pathway on the osteogenic differentiation of PDLSCs under the influence of TNF-α.
PDLSCs were obtained through enzyme digestion and tissue block method. The expression levels of stem cell surface markers CD105, CD90, CD146, CD45, and CD31 were detected by fluorescence activated cell sorter (FACS). PDLSCs were divided into experimental (10 ng·mL⁻¹ TNF-α) and control groups (0 ng·mL⁻¹ TNF-α). The proliferation ability of PDLSCs was detected using cell counting kit-8 (CCK-8). The effect of TNF-α on the osteogenic ability of PDLSCs were tested by measuring alkaline phosphatase (ALP) activity and conducting alizarin red staining and quantitative real-time polymerase chain reaction (PCR). We tested Notch signal pathway receptors Notch1, Notch2, ligand JAG1, JGA2, and downstream gene Hes-1. Changes in DLL1 expression were detected by quantitative real-time PCR.
FACS profiling showed that PDLSCs were strongly positive for CD105, CD90, and CD146 but negative for CD45 and CD31. CCK-8 results showed that TNF-α could promote the proliferation of PDLSCs (P<0.05). ALP activity in the experimental group was lower than that in the control group (P<0.05). Alizarin red staining showed that the experimental group had decreased mineralized nodules as compared with the control group. Quantitative real-time PCR results showed that the mRNA expression of osteogenic marker genes cementum attachment protein (CAP), osteopontin (OPN), and Runt-related transcription factor 2 (Runx2) significantly decreased in the experimental group as compared with those in the control group (P<0.05). The expression levels of Notch1, Notch2, JAG1, JGA2 and Hes-1 were significantly decreased (P<0.05), whereas those of Notch3 and DLL1 were increased in Notch signaling pathway-related molecules (P<0.05).
TNF-α can promote PDLSCs proliferation and inhibit bone differentiation and Notch signaling pathway expression, indicating that the Notch signaling pathway regulates PDLSCs osteogenic differentiation.
评估肿瘤坏死因子-α（TNF-α）对牙周膜干细胞（PDLSCs）成骨分化及Notch信号通路的影响，并探讨在TNF-α影响下Notch信号通路对PDLSCs成骨分化的调控作用。
通过酶消化法和组织块法获取PDLSCs。采用荧光激活细胞分选仪（FACS）检测干细胞表面标志物CD105、CD90、CD146、CD45和CD31的表达水平。将PDLSCs分为实验组（10 ng·mL⁻¹ TNF-α）和对照组（0 ng·mL⁻¹ TNF-α）。使用细胞计数试剂盒-8（CCK-8）检测PDLSCs的增殖能力。通过测量碱性磷酸酶（ALP）活性、进行茜素红染色和定量实时聚合酶链反应（PCR）来检测TNF-α对PDLSCs成骨能力的影响。我们检测了Notch信号通路受体Notch1、Notch2、配体JAG1、JGA2和下游基因Hes-1。通过定量实时PCR检测DLL1表达的变化。
FACS分析显示，PDLSCs对CD105、CD90和CD146呈强阳性，而对CD45和CD31呈阴性。CCK-8结果显示，TNF-α可促进PDLSCs的增殖（P<0.05）。实验组的ALP活性低于对照组（P<0.05）。茜素红染色显示，与对照组相比，实验组矿化结节减少。定量实时PCR结果显示，与对照组相比，实验组中成骨标志物基因牙骨质附着蛋白（CAP）、骨桥蛋白（OPN）和Runt相关转录因子2（Runx2）的mRNA表达显著降低（P<0.05）。Notch信号通路相关分子中，Notch1、Notch2、JAG1、JGA2和Hes-1的表达水平显著降低（P<0.05），而Notch3和DLL1的表达水平升高（P<0.05）。
TNF-α可促进PDLSCs增殖，抑制其骨分化及Notch信号通路表达，表明Notch信号通路调控PDLSCs的成骨分化。
