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  • Title: [Naked cuticle homolog 2 positively regulates the osteogenic differentiation of rat dental follicle cells].
    Author: Hou YL, Ling JQ, Chen CC, Quan JJ, Du Y.
    Journal: Zhonghua Kou Qiang Yi Xue Za Zhi; 2017 Jul 09; 52(7):432-438. PubMed ID: 29972908.
    Abstract:
    Objective: To examine the expression of naked cuticle homolog 2 (Nkd2) in the process of root development and osteogenic differentiation of dental follicle cells of rat (rDFC), in order to explore the molecular mechanisms of Nkd2 on the osteoblast differentiation of rDFCs. Methods: Immunohistochemical analysis was used to detect the expression of Nkd2 in the base dental follicle of the mandibular first molar of rat at 1, 3, 5, 7, 9, 11 and 13 days postnatal. Mineralization nodule formation of rDFCs was detected by alizarin red staining and cetylpyridine. The change of Nkd2 during osteogenic differentiation of rDFCs was evaluated by Western blotting and the associations between Nkd2 and osteogenic cytokines of alkaline phosphatase (ALP), Runt-related transcription factor-2 (RUNX2) and osteocalcin (OCN) were examined. The rDFCs were transfected with small interfering RNA (siRNA) to knock down the expression of Nkd2 and Western blotting and quantitative real-time PCR (qPCR) were adopted to explore the effects of Nkd2 on osteogenic differentiation by detecting variations of Nkd2 and osteogenic factors ALP, RUNX2, OCN among silencing group (Si), negative control RNA group (Nc) and mock control group (Mock), respectively. Results: The expression of Nkd2 in the base dental follicle of the mandibular first molar of rat was time dependent. Mineralization nodules of rDFCs and absorbance of cetylpyridine after osteogenic induction increased gradually (the absorbances of cetylpyridine were 0 week: 0.017±0.005, 1 week: 0.702±0.044, 2 weeks: 1.812±0.531, 3 weeks: 2.767±0.253, respectively). Results of Western blotting showed that Nkd2 (1.60±0.23) of mineralization group was significantly higher than that of control group (1) (P<0.05) at the early stage of osteogenic differentiation along with the expression of other osteogenic factors. The protein and mRNA of Nkd2 and osteogenic factors were significantly decreased in Si group compared with Nc and Mock groups (P<0.05), and no changes between Nc and Mock groups were observed. The changes of protein in Si, Nc and Mock groups were Nkd2: 0.42±0.10, 1.12±0.07, 1, ALP: 0.70±0.15, 1.11±0.14, 1, RUNX2: 0.58±0.08, 0.93±0.08, 1 and OCN: 0.64±0.06, 0.99±0.02, 1, respectively. The mRNA variances in Si, Nc and Mock groups were Nkd2: 0.39±0.05, 0.96±0.10, 1, ALP: 0.15±0.13, 1.01±0.07, 1, RUNX2: 0.39±0.31, 0.97±0.13, 1, OCN: 0.17±0.08, 1.08±0.21, 1, respectively. Conclusions: Nkd2 participates in the root development process in rat and may acts as a positive role in the early stage of osteogenic differentiation of rDFCs in rat. 目的： 检测裸露角质蛋白同源物2（naked cuticle homolog 2，Nkd2）在大鼠牙根形成和牙囊细胞成骨向分化过程中的表达变化，为探讨Nkd2在牙囊细胞成骨向分化中的具体分子机制提供实验基础。 方法： 免疫组化法检测Nkd2在SD大鼠出生后1、3、5、7、9、11及13 d下颌第一磨牙牙胚近基底侧牙囊组织中的表达。茜素红染色和十六烷基吡啶观察分析大鼠牙囊细胞（dental follicle cells of rat，rDFC）矿化诱导1、2、3周钙化结节形成情况。蛋白质印迹法分析rDFC矿化诱导1、2、3周Nkd2蛋白表达变化及其与成骨因子碱性磷酸酶（alkaline phosphatase，ALP）、Runt相关转录因子2（Runt-related transcription factor-2，RUNX2）和骨钙蛋白的关系。小干扰RNA（small interfering RNA，siRNA）沉默rDFC中Nkd2（siRNA干扰组，Si组），经矿化诱导1周，蛋白质印迹法和实时荧光定量PCR（quantitative real-time PCR，qPCR）检测Si组、阴性对照组（阴性对照RNA组，negative control RNA group，Nc组）和空白对照组（mock control group，Mock组）Nkd2及其mRNA与ALP、RUNX2和骨钙蛋白的表达变化。 结果： 免疫组化结果显示，大鼠牙根形成中Nkd2在下颌第一磨牙牙胚基底侧牙囊组织中的表达呈现时间差异。随rDFC成骨诱导时间增加，茜素红染色矿化结节逐渐增多，十六烷基吡啶吸光度值逐渐增高（0、1、2、和3周吸光度值分别为0.017±0.005、0.702±0.044、1.812±0.531及2.767±0.253）。蛋白质印迹法结果显示，矿化诱导早期矿化组Nkd2（1.60±0.23）较对照组（1）表达显著上调（P<0.05），Nkd2表达趋势与成骨相关因子表达趋势一致。siRNA干扰rDFC矿化诱导1周后Si组较Nc、Mock组Nkd2和成骨因子在蛋白和mRNA水平显著降低（P<0.05），Nc与Mock组Nkd2和成骨因子表达差异无统计学意义（P>0.05）。Si、Nc和Mock组蛋白相对表达量分别为Nkd2：0.42±0.10、1.12±0.07、1；ALP：0.70±0.15、1.11±0.14、1；RUNX2：0.58±0.08、0.93±0.08、1；骨钙蛋白：0.64±0.06、0.99±0.02、1；Si、Nc和Mock组mRNA相对表达量分别为Nkd2：0.39±0.05、0.96±0.10、1；ALP：0.15±0.13、1.01±0.07、1；RUNX2：0.39±0.31、0.97±0.13、1；骨钙蛋白：0.17±0.08、1.08±0.21、1。 结论： 大鼠牙根形成过程中Nkd2参与了牙囊组织的分化，并正向调控大鼠牙囊细胞的早期成骨向分化。.
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