中国实用口腔科杂志

中国实用口腔科杂志 ›› 2024, Vol. 17 ›› Issue (5): 568-573.DOI: 10.19538/j.kq.2024.05.011

• 论著 • 上一篇    下一篇

神经纤毛蛋白-1在前成骨细胞成骨分化过程中及小鼠胫骨组织中的表达研究

姚澳康，李宗谕，杨心月，杨    谛，仇丽鸿，于雅琼   

  1. 中国医科大学附属口腔医院牙体牙髓病科，辽宁省口腔疾病重点实验室，辽宁 沈阳 110002
  • 出版日期:2024-09-30 发布日期:2024-09-30
  • 基金资助:
    国家自然科学基金青年科学基金项目（81900991）

  • Online:2024-09-30 Published:2024-09-30

摘要： 目的    探究神经纤毛蛋白-1（neuropilin 1，NRP1）在前成骨细胞MC3T3-E1成骨分化过程中的mRNA和蛋白表达特征，以及其在小鼠胫骨组织中的表达情况，为临床应用信号素3A/NRP1信号轴治疗慢性根尖周炎区骨吸收提供实验基础。方法    研究于2023年4—6月在中国医科大学附属口腔医院中心实验室进行。成骨分化诱导培养MC3T3-E1，并根据成骨分化时间分组。采用Western blot检测对照组（0 d）和分化1、3、5、7、10、14、20 d组的NRP1蛋白表达情况；采用qRT⁃PCR检测对照组和分化1、3、5、7、10、14 d组的NRP1 mRNA表达情况；对照组和成骨分化3、5 d组细胞进行免疫荧光染色，采用激光共聚焦显微镜观察成骨分化前期NRP1亚细胞定位。将3只4日龄小鼠共6根胫骨均分为NRP1组和阴性对照IgG组，并进行免疫荧光染色，采用倒置荧光显微镜观察NRP1在小鼠胫骨组织中的表达情况。结果    各组NRP1蛋白及mRNA的相对表达量比较，差异均有统计学意义（F值分别为48600.000、60.030，均P < 0.001）；且分化7、10 d组的NRP1蛋白相对表达量较其他时间组高，分化10 d组的NRP1 mRNA相对表达量最高，差异均有统计学意义（均P < 0.05）。在Western blot检测中NRP1主条带下方观察到分泌型NRP1（soluble NRP1，sNRP1）副条带。对照组和分化3、5 d组中，NRP1均在细胞质中表达，其亚细胞定位表现为围绕细胞核周围。NRP1在小鼠胫骨干骺端表达，且成骨区成骨细胞内表达广泛。结论    NRP1参与前成骨细胞成骨分化过程，成骨分化前期主要在细胞质中表达，且参与小鼠胫骨形成。

关键词: 神经纤毛蛋白-1, 前成骨细胞, 成骨分化

Abstract: Objective    To investigate the expression of neuropilin 1（NRP1）mRNA and protein during the osteogenic differentiation of MC3T3-E1（a preosteoblastic cell line），as well as the expression of NRP1 in the mouse tibial tissue，in order to provide an experimental foundation for the clinical application of Semaphorin 3A/NRP1 signaling pathway in the treatment of bone resorption in chronic periapical inflammation. Methods    This study was conducted from April to June 2023 in the Central Laboratory of the Stomatological Hospital of China Medical University. MC3T3-E1 cells were cultured for osteogenic differentiation induction and grouped based on the duration of osteogenic differentiation. Western blot analysis was used to detect the expression of NRP1 protein in the control group（0 days）and in the differentiated groups at 1，3，5，7，10，14，and 20 days. Quantitative real-time PCR（qRT-PCR）was used to detect the expression of NRP1 mRNA in the control group and in the differentiation groups at 1，3，5，7，10，and 14 days. Immunofluorescence staining was performed on cells in the control group and in the differentiation groups at 3 and 5 days，and subcellular localization of NRP1 during the early stage of osteogenic differentiation was observed using confocal laser scanning microscopy. Six tibiae from three 4-day-old mice were equally divided into NRP1 and negative control IgG groups，immunofluorescence staining was performed，and an inverted fluorescence microscope was used to demonstrate NRP1 expression in mouse tibial tissues. Results    The relative expression levels of NRP1 protein and mRNA were compared among the groups，and significant differences were found（F values were 48600.000 and 60.030，respectively，both P < 0.001）. Specifically，the relative expression levels of NRP1 protein in the 7-day and 10-day differentiation groups were higher than those in other time groups，and the relative expression level of NRP1 mRNA in the 10-day differentiation group was the highest，with statistically significant differences observed（all P < 0.05）. In the Western blot analysis，a secondary band corresponding to the secreted form of NRP1（soluble NRP1，sNRP1）was observed below the main NRP1 band. In the control group and the 3 and 5-day differentiation groups，NRP1 was expressed in the cytoplasm，with its subcellular localization surrounding the nucleus. NRP1 was expressed in the metaphyseal region of the mouse tibia，with widespread expression in osteoblasts within the bone-forming areas. Conclusion    NRP1 participates in the osteogenic differentiation process of MC3T3-E1，primarily expressed in the cytoplasm during the early stage of osteogenic differentiation，and contributes to the formation of the mouse tibia.

Key words: neuropilin 1, preosteoblastic cells, osteogenic differentiation