Yi X S, Li Y Z, Mo S Y, Xie Q F
Department of Implantology, Peking University School and Hospital of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China.
Department ofProsthodontics, Peking University School and Hospital of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China(Li Yuzhou is now working on the Department of Prosthodontics, Stomatological Hospital of Chongqing Medical University & Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences & Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education, Chongqing 401120, China).
Zhonghua Kou Qiang Yi Xue Za Zhi. 2020 Jun 9;55(6):394-401. doi: 10.3760/cma.j.cn112144-20191210-00444.
To choose a suitable efficient concentration of adenosine triphosphate (ATP) which can induce human dental pulp cells (HDPC) differentiate into odontoblast successfully, and explore the role of this concentration of ATP in dentin regeneration . HDPC were treated with different concentrations (0, 10, 400, 600, 800 μmol/L) of ATP. Then cell counting kit-8 (CCK-8), quantitative real-time PCR, and Western blotting were used to detect the cell proliferation and the expressions of odontoblastic differentiation related markers, dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP). Alizarin red S staining experiment was used to analyze the effect of ATP on the mineralization ability of HDPC. By the above experiments, the suitable effective concentration of ATP was chosen to pretreat the HDPC for 7 days, then cells were seeded on gelfoam, inserted into the root canal fragment, and subsequently transplanted into the subcutaneous space on the back of immunodeficient mice, after three months, the samples were stained with HE for histological analysis. The CCK-8 results in 5 d showed that 10 μmol/L ATP obviously promoted the proliferation of HDPC, while the 600 and 800 μmol/L ATP apparently inhibited the HDPC proliferation, however, the proliferation in 800 μmol/L ATP group was lower than that of 600 μmol/L ATP group (0.05). qPCR and Western blotting results showed that the 600 and 800 μmol/L ATP significantly up-regulated the DMP1 and DSPP expressions (0.05), furthermore, there was no significant difference in the two groups, but no changes were found in other groups (0.05). After 21 days of culturing, there were obvious mineralization nodules in 600 and 800 μmol/L ATP groups, but no mineralization nodules in other groups. Quantitative analysis of the staining results showed the value in 0, 10, 400, 600, and 800 μmol/L ATP groups were respectively 1.05±0.15, 1.11±0.23, 1.15±0.17, 3.65±0.30, and 3.40±0.43, and the value in 600 and 800 μmol/L ATP groups were higher than those of other groups; however, there was no difference in 600 and 800 μmol/L ATP groups. The histological analysis showed that 600 μmol/L ATP could induce the HDPC differentiate into dentin-like structure in the root canal fragment. Therefore, the suitable effective concentration of ATP is 600 μmol/L, which could induce HDPC differentiate into odontoblast-like cells, and form the dentin-like structure .
为选择一种合适的能成功诱导人牙髓细胞（HDPC）分化为成牙本质细胞的高效三磷酸腺苷（ATP）浓度，并探讨该浓度的ATP在牙本质再生中的作用。用不同浓度（0、10、400、600、800μmol/L）的ATP处理HDPC。然后使用细胞计数试剂盒-8（CCK-8）、定量实时聚合酶链反应和蛋白质免疫印迹法检测细胞增殖以及成牙本质细胞分化相关标志物牙本质基质蛋白1（DMP1）和牙本质涎磷蛋白（DSPP）的表达。茜素红S染色实验用于分析ATP对HDPC矿化能力的影响。通过上述实验，选择合适的有效ATP浓度对HDPC预处理7天，然后将细胞接种于明胶海绵上，插入根管片段，随后移植到免疫缺陷小鼠背部的皮下空间，三个月后，样本用苏木精-伊红染色进行组织学分析。CCK-8结果显示，在第5天时，10μmol/L的ATP明显促进HDPC增殖，而600和800μmol/L的ATP明显抑制HDPC增殖，然而，800μmol/L ATP组的增殖低于600μmol/L ATP组（P<0.05）。定量聚合酶链反应和蛋白质免疫印迹结果显示，600和800μmol/L的ATP显著上调DMP1和DSPP的表达（P<0.05），此外，两组之间无显著差异，但其他组未发现变化（P<0.05）。培养21天后，600和800μmol/L ATP组有明显的矿化结节，而其他组没有。染色结果的定量分析显示，0、10、400、600和800μmol/L ATP组的吸光度值分别为1.05±0.15、1.11±0.23、1.15±0.17、3.65±0.30和3.40±0.43，600和800μmol/L ATP组的吸光度值高于其他组；然而，600和800μmol/L ATP组之间没有差异。组织学分析表明，600μmol/L的ATP可诱导HDPC在根管片段中分化为牙本质样结构。因此，合适的有效ATP浓度为600μmol/L，其可诱导HDPC分化为成牙本质样细胞，并形成牙本质样结构。
