南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (8): 1339-1344.doi: 10.12122/j.issn.1673-4254.2023.08.10

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脂质运载蛋白2自限性抑制间充质干细胞的成骨细胞分化

陈梓锋，李胜发，张祐鸣，杨婉雯，王 婷   

  1. 广州市番禺区中心医院创伤骨科，广东 广州 511400；南方医科大学珠江医院临床研究中心，广东 广州 510260；南方医科大学基础医学院细胞生物学，广东 广州 510515
  • 出版日期:2023-08-20 发布日期:2023-09-11

Lipocalin 2 induces self- limited inhibition of osteoblast differentiation of mesenchymal stem cells

CHEN Zifeng, LI Shengfa, ZHANG Youming, YANG Wanwen, WANG Ting   

  1. Department of Orthopedics, Panyu Central Hospital, Guangzhou 511400, China; Department of Clinical Research Center, Zhujiang Hospital, Southern Medical University, Guangzhou 510260, China; Department of Cell Biology, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
  • Online:2023-08-20 Published:2023-09-11

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摘要： 目的 探究脂质运载蛋白2（Lcn2）在骨发育和老年性骨质疏松的作用。方法 C3H10T1/2 MSC细胞用成骨诱导液诱导3 d或不作处理（control），收集细胞核提取染色质，以H3K27A和H3K9Me3抗体进行免疫沉淀；ChIP-seq分析了诱导成骨分化前后C3H10T1/2间充质干细胞Lcn2基因座位的修饰组蛋白结合情况；ROSE软件（利用H3K27AC信号计算）显示成骨诱导后出现超级增强子；RNA-seq结果证实Lcn2在C3H10T1/2成骨诱导后上调的倍数在所有基因中位列第4；逆转录-实时定量PCR结果用于证实Lcn2的mRNA水平在C3H10T1/2成骨诱导后上调；蛋白质组学分析了16月龄C57BL/6J雄鼠相对于3月龄雄鼠股骨远端总蛋白表达谱的差异。iTRAQ-MS/MS用于分析，16月龄的老年雄鼠的松质骨中Lcn2蛋白表达相对于3月龄雄鼠的差异；ELISA检测3月龄和16月龄老年雄鼠血清中Lcn2含量；用50、100、200、500 ng/mL浓度的Lcn2重组蛋白处理C3H10T1/2后以ALP检测成骨分化能力，以Westem blot检测早期成骨转录因子OSX和晚期成骨指标OCN。结果 iTRAQ-MS/MS结果显示，16月龄的老年雄鼠的松质骨中Lcn2蛋白表达相对于3月龄雄鼠明显上调（P<0.01）。与此对应，ELISA结果示16月龄老年雄鼠血清中Lcn2含量比3月龄对照（control）雄鼠明显升高（P<0.01）。ALP染色结果和定量结果显示加入100、200、500 ng/mL浓度Lcn2 RP后ALP表达减弱（P<0.05，P<0.01，P<0.01）。Westem blot定量结果显示OSX在100、200、500 ng/mL浓度的Lcn2 RP组中的表达减低，呈剂量依赖性（P<0.05）。Westem blot定量结果显示OCN在100、200、500 ng/mL浓度的Lcn2 RP组中的表达减低，呈剂量依赖性（P<0.05，P<0.01，P<0.001）。ROSE软件（利用H3K27AC信号计算）显示成骨诱导后Lcn2基因区域出现超级增强子。RNA-seq结果证实Lcn2在C3H10T1/2成骨诱导后上调的倍数在所有基因中位列第4。C3H10T1/2成骨诱导后上调倍数最高的Zbtb16基因区域也在成骨诱导后出现超级增强子。逆转录-实时定量PCR进一步证实了Lcn2的mRNA水平在成骨诱导后急剧升高（P<0.001）。结论 Lcn2是间充质干细胞（MSCs）正常成骨分化所必须的，但在衰老时过度表达和分泌以自限性抑制MSCs的成骨分化。

关键词: 脂质运载蛋白2；老年性骨质疏松；间充质干细胞；成骨分化

Abstract: Objective To explore the role of lipocalin 2 (Lcn2) in bone development and senile osteoporosis. Methods Chromatin immunoprecipitation with H3K27AC and H3K9Me3 antibodies coupled with massively parallel sequencing (ChIP- Seq) was performed to analyze the changes in binding of modified histones at the Lcn2 gene locus in C3H10T1/2 mesenchymal stem cells (MSCs) induced with osteogenic induction medium for 3 days. ITRAQ-MS/MS and ELISA were used to compare Lcn2 protein expressions in the cancellous bone and the serum between 16- and 3-month-old male mice. The effect of treatment with recombinant Lcn2 protein on osteogenic differentiation of C3H10T1/2 cells was evaluated by detecting changes in ALP expression, and Western blotting was performed to examine the changes in OSX and OCN expression. Results The expression of Lcn2 protein in the cancellous bone and its serum levels were significantly higher in 16-month-old than in 3-month-old male mice (P<0.01). In C3H10T1/2 cells, ALP expression level decreased significantly after treatment with recombinant Lcn2 protein (P<0.05) accompanied by lowered protein expressions of OSX and OCN (P<0.05). Analysis with ROSE software revealed a super enhancer in the Lcn2 gene region of C3H10T1/2 cells after osteogenesis induction. RNA-seq results confirmed that Lcn2 was among the top 4 up- regulated genes in C3H10T1/2 cells following osteogenic induction, and a super enhancer was also detected in Zbtb16 gene, which showed the highest up- regulation after osteogenic induction. Real- time quantitative PCR further confirmed that the mRNA level of Lcn2 increased sharply in C3H10T1/2 cells after osteogenic induction (P<0.001). Conclusion Lcn2 is essential for normal osteogenic differentiation of MSCs, but its overexpression and excessive secretion in aging induce self-limited inhibition of osteogenic differentiation of MSCs.

Key words: lipocalin 2; senile osteoporosis; mesenchymal stem cells; osteogenic differentiation