中国组织工程研究

中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (18): 3741-3746.doi: 10.12307/2025.666

• 骨组织构建 bone tissue construction •    下一篇

初级纤毛介导成骨前体细胞系MC3T3-E1传代衰老减低成骨分化能力

高  丰1，王纪亮1，王洪波1，杨永胜1，刘  源1，付  苏2   

  1. 1内蒙古自治区中医医院，内蒙古自治区呼和浩特市  010000；2郑州大学第一附属医院骨科，河南省郑州市  450000
  • 收稿日期:2024-04-02 接受日期:2024-08-12 出版日期:2025-06-28 发布日期:2024-11-27
  • 通讯作者: 高丰，博士，主治医师，内蒙古自治区中医医院，内蒙古自治区呼和浩特市 010000
  • 作者简介:高丰，男，1986年生，内蒙古自治区呼和浩特市人，汉族，2019年南方医科大学毕业，博士，主治医师，主要从事骨质疏松研究。
  • 基金资助:
    国家博士后自然科学面上基金(2020M682359)，项目负责人：付苏

Passage-associated senescence decreases osteogenic activity of MC3T3-E1 cells via primary cilia 

Gao Feng1, Wang Jiliang1, Wang Hongbo1, Yang Yongsheng1, Liu Yuan1, Fu Su2   

  1. 1Inner Mongolia Hospital of Traditional Chinese Medicine, Hohhot 010000, Inner Mongolia Autonomous Region, China; 2Department of Orthopedics, First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, Henan Province, China
  • Received:2024-04-02 Accepted:2024-08-12 Online:2025-06-28 Published:2024-11-27
  • Contact: Gao Feng, Inner Mongolia Hospital of Traditional Chinese Medicine, Hohhot 010000, Inner Mongolia Autonomous Region, China
  • About author:Gao Feng, MD, Attending physician, Inner Mongolia Hospital of Traditional Chinese Medicine, Hohhot 010000, Inner Mongolia Autonomous Region, China
  • Supported by:
    National Postdoctoral Natural Science Foundation, No. 2020M682359 (to FS)

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摘要：


文题释义：
初级纤毛：真核细胞的长形力学感受性细胞器，介导力学信号传导及多种信号通路。
鞭毛转运系统：初级纤毛的核心轴丝动力系统，维持初级纤毛长度的动态平衡。

背景：在大段骨缺损修复中，由于种子细胞传代等多种因素可引起成骨类细胞衰老，造成组织工程骨植入体内后成骨分化活性降低。近年来，初级纤毛介导细胞衰老的机制已被广泛研究，但“传代衰老-成骨活性减低”的初级纤毛相关机制尚未完全明了。
目的：探讨初级纤毛调控成骨性MC3T3-E1细胞传代衰老的可能机制。
方法：成骨前体细胞系MC3T3-E1细胞传代至第10代(早期传代)及第40代(晚期传代)，同时使用siRNA沉默IFT88阻碍初级纤毛生成，即将细胞分为第10代组、第40代组、第10代+siRNA IFT88-组、第40代+siRNA IFT88-组。各组细胞成骨诱导3 d，采用RT-PCR和Western blot检测衰老标志物CDKN2A(P16)的表达、成骨活性标志物骨形态发生蛋白2和碱性磷酸酶的表达、Hedgehog通路IHH的表达；茜素红染色和初级纤毛免疫荧光染色分析初级纤毛形态；对初级纤毛阳性率与IHH、骨形态发生蛋白2的表达进行Spearman相关性分析。
结果与结论：①成骨诱导3 d，第40代组MC3T3-E1细胞的CDKN2A(P16)表达显著高于第10代组，而在siRNA IFT88-干预后差异消失；②第10代组的初级纤毛细胞阳性率高于第40代组，siRNA IFT88-则显著性抑制第10代与第40代细胞的初级纤毛表达；③第10代组MC3T3-E1细胞的碱性磷酸酶及骨形态发生蛋白2的转录活性和蛋白表达均高于第40代组，通过siRNA抑制初级纤毛表达后，上述差异减低或者消失；④初级纤毛细胞阳性率与IHH蛋白表达及骨形态发生蛋白2蛋白表达呈正相关。结果表明：初级纤毛介导了成骨性MC3T3-E1细胞的传代衰老，可调控成骨分化能力。
https://orcid.org/0009-0008-8312-0484(高丰)

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: MC3T3-E1细胞, 初级纤毛, 衰老, 成骨分化, 传代, 骨形态发生蛋白2, Hedgehog通路, 组织工程细胞

Abstract: BACKGROUND: In the repair of large bone defects, a variety of factors such as seed cell passaging can cause senescence of osteoblasts, leading to a reduction in osteogenic differentiation activity after implantation of tissue-engineered bone. In recent years, a novel mechanism involving primary cilia in cell senescence has been widely studied, but the primary cilia-related mechanism of "passage senescence - reduced osteogenic activity" is not fully understood.
OBJECTIVE: To explore the possible mechanisms by which primary cilia regulate the senescence of MC3T3-E1 cells.
METHODS: The osteoblast precursor cell lines MC3T3-E1 were passaged to 10th generation cells (early passage) and 40th generation cells (late passage). siRNA was used to silence IFT88 to inhibit primary cilia formation. The cells were than grouped into passage 10 group, passage 40 group, passage 10+siRNA IFT88 group, and passage 40+siRNA IFT88 group. RT-PCR and western blot assays were used to detect the expression of the aging marker P16 (CDKN2A), the osteogenic activity markers bone morphogenetic protein 2 and alkaline phosphatase, and the Hedgehog pathway IHH expression. Alizarin red staining and primary cilia immunofluorescence staining were performed. Spearman correlation analysis was conducted to analyze primary cilia positive rate and IHH and bone morphogenetic protein 2 expression. 
RESULTS AND CONCLUSION: (1) The expression of CDKN2A (P16) in the passage 10 group was significantly higher than in the passage 40 group, but the difference disappeared after siRNA IFT88 intervention. (2) Meanwhile, the positive rate of primary cilia cells in the passage 10 group were higher than in the passage 40 group, while siRNA IFT88- significantly inhibited the expression of primary cilia in both passage 10 and passage 40 cells. (3) The transcriptional activity and protein expression of bone morphogenetic protein 2 and alkaline phosphatase in the passage 10 group were higher than those in the passage 40 group. After inhibiting the expression of primary cilia with siRNA, the above differences were reduced or disappeared. (4) The positive rate of primary cilia cells was correlated with IHH and bone morphogenetic protein 2 protein expression. To conclude, primary cilia mediate the replicative senescence of osteogenic MC3T3-E1 cells and regulate osteogenic differentiation ability. 

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: MC3T3-E1 cells, primary cilia, senescence, osteogenic differentiation, passage, bone morphogenetic protein 2, Hedgehog pathway, tissue- engineered cells

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