Hou Y L, Ling J Q, Chen C C, Quan J J, Du Y
Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University & Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China.
Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University & Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China [Present adress: Department of Stomatology, Shenzhen Children's Hosptial, Shenzhen Guangdong 518033, China].
Zhonghua Kou Qiang Yi Xue Za Zhi. 2017 Jul 9;52(7):432-438. doi: 10.3760/cma.j.issn.1002-0098.2017.07.008.
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. 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. 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) (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 (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. 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.
为研究裸角质膜同源物2（Nkd2）在大鼠牙囊细胞（rDFC）牙根发育和成骨分化过程中的表达，以探讨Nkd2对rDFCs成骨细胞分化的分子机制。采用免疫组织化学分析法检测出生后1、3、5、7、9、11和13天大鼠下颌第一磨牙牙囊基部Nkd2的表达。通过茜素红染色和十六烷基吡啶检测rDFCs矿化结节的形成。采用蛋白质免疫印迹法评估rDFCs成骨分化过程中Nkd2的变化，并检测Nkd2与碱性磷酸酶（ALP）、Runt相关转录因子2（RUNX2）和骨钙素（OCN）等成骨细胞因子之间的关联。用小干扰RNA（siRNA）转染rDFCs以敲低Nkd2的表达，分别通过检测沉默组（Si）、阴性对照RNA组（Nc）和模拟对照组（Mock）中Nkd2及成骨因子ALP、RUNX2、OCN的变化，采用蛋白质免疫印迹法和定量实时聚合酶链反应（qPCR）探讨Nkd2对成骨分化的影响。大鼠下颌第一磨牙牙囊基部Nkd2的表达呈时间依赖性。成骨诱导后rDFCs矿化结节及十六烷基吡啶吸光度逐渐增加（十六烷基吡啶吸光度分别为0周：0.017±0.005，1周：0.702±0.044，2周：1.812±0.531，3周：2.767±0.253）。蛋白质免疫印迹结果显示，在成骨分化早期，矿化组的Nkd2（1.60±0.23）显著高于对照组（1）（P<0.05），同时其他成骨因子也表达。与Nc组和Mock组相比，Si组中Nkd2及成骨因子的蛋白质和mRNA水平显著降低（P<0.05），Nc组和Mock组之间未观察到变化。Si、Nc和Mock组蛋白质变化分别为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；OCN：0.64±0.06，0.99±0.02，1。Si、Nc和Mock组mRNA变化分别为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；OCN：0.17±0.08，1.08±0.21，1。Nkd2参与大鼠牙根发育过程，可能在大鼠rDFCs成骨分化早期起正向作用。
