中国组织工程研究

中国组织工程研究 ›› 2026, Vol. 30 ›› Issue (7): 1649-1657.doi: 10.12307/2026.086

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

高糖微环境中程序性细胞死亡受体1抑制大鼠骨髓间充质干细胞的成骨分化

韩念荣，黄异飞，艾克热木·吾斯曼，刘岩路，胡  炜   

  1. 新疆医科大学附属中医医院脊柱二科，新疆维吾尔自治区乌鲁木齐市   830000
  • 收稿日期:2025-02-06 修回日期:2025-05-27 接受日期:2025-06-20 出版日期:2026-03-08 发布日期:2025-08-18
  • 通讯作者: 胡炜，主任医师，新疆医科大学附属中医医院脊柱二科，新疆维吾尔自治区乌鲁木齐市   830000
  • 作者简介:韩念荣，男，1992 年生，河南省驻马店市人，汉族，新疆医科大学在读博士，主治医师，主要从事中西医结合脊柱外科方面的研究。
  • 基金资助:
    新疆维吾尔自治区自然科学基金—杰出青年科学基金(2022D01E29)，项目负责人：胡炜

Programmed cell death receptor-1 suppresses osteogenic differentiation of rat bone marrow mesenchymal stem cells in a high-glucose microenvironment 

Han Nianrong, Huang Yifei, Akram · Osman, Liu Yanlu, Hu Wei    

  1. Department of Spine II, Hospital of Traditional Chinese Medicine Affiliated to Xinjiang Medical University, Urumqi 830000, Xinjiang Uygur Autonomous Region, China 
  • Received:2025-02-06 Revised:2025-05-27 Accepted:2025-06-20 Online:2026-03-08 Published:2025-08-18
  • Contact: Hu Wei, Chief physician, Department of Spine II, Hospital of Traditional Chinese Medicine Affiliated to Xinjiang Medical University, Urumqi 830000, Xinjiang Uygur Autonomous Region, China
  • About author:Han Nianrong, Doctoral candidate, Attending physician, Department of Spine II, Hospital of Traditional Chinese Medicine Affiliated to Xinjiang Medical University, Urumqi 830000, Xinjiang Uygur Autonomous Region, China
  • Supported by:
    Xinjiang Uygur Autonomous Region Natural Science Foundation - Outstanding Young Scientist Fund, No. 2022D01E29 (to HW) 

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摘要：

文题释义：

骨髓间充质干细胞：作为骨髓内的多能性细胞，具备分化为成骨细胞的能力，在骨骼的形成、维系以及重塑过程中扮演着关键角色。 
神经前体细胞发育下调蛋白4：是E3泛素连接酶的一部分，与骨相关的生物过程有关，可以通过调控成骨相关信号通路(如BMP/Smad、Wnt/β-catenin、PTEN/PI3K/Akt通路)和转录因子(Runt相关转录因子2、Osterix)的活性，参与成骨分化。

摘要
背景：程序性细胞死亡受体1和神经前体细胞发育下调蛋白4参与调控成骨细胞的分化，但这两者间的相互作用及调控机制仍需进一步研究阐明。
目的：探讨高糖环境中程序性细胞死亡受体1调控神经前体细胞发育下调蛋白4影响大鼠骨髓间充质干细胞成骨分化的机制。
方法：①采用免疫沉淀-质谱联用技术检测程序性细胞死亡受体1的交互蛋白，采用免疫共沉淀验证程序性细胞死亡受体1与神经前体细胞发育下调蛋白4的交互作用，采用免疫荧光检测程序性细胞死亡受体1和神经前体细胞发育下调蛋白4的定位。②将第3代大鼠骨髓间充质干细胞随机分为正常糖组(5.6 mmol/L)、高糖组(30 mmol/L)、程序性细胞死亡受体1敲减空载组、程序性细胞死亡受体1敲减组、程序性细胞死亡受体1过表达空载组、程序性细胞死亡受体1过表达组，采用Western blot检测神经前体细胞发育下调蛋白4的蛋白表达。③将第3代大鼠骨髓间充质干细胞随机分为正常糖组(5.6 mmol/L)、高糖组(30 mmol/L)、神经前体细胞发育下调蛋白4敲减组，采用qRT-PCR检测神经前体细胞发育下调蛋白4及成骨标志物OSX、Runt相关转录因子2 mRNA表达，茜素红S染色和碱性磷酸酶染色评估成骨分化能力，Western blot检测Runt相关转录因子2、OSX、AKT、PI3K、p-PI3K、p-AKT蛋白表达。④随后在程序性细胞死亡受体1过表达的同时进行神经前体细胞发育下调蛋白4敲减处理，开展回复实验，观察细胞成骨分化能力变化。

结果与结论：①免疫沉淀-质谱联用技术、免疫共沉淀和免疫荧光实验显示神经前体细胞发育下调蛋白4是程序性细胞死亡受体1的交互蛋白，程序性细胞死亡受体1与神经前体细胞发育下调蛋白4共定位；②神经前体细胞发育下调蛋白4敲减组程序性细胞死亡受体1的mRNA与蛋白表达水平较高糖组降低(P < 0.05)；③神经前体细胞发育下调蛋白4敲减组大鼠骨髓间充质干细胞的成骨分化增强，激活PI3K/AKT通路；④程序性细胞死亡受体1过表达+神经前体细胞发育下调蛋白4敲减组的成骨细胞分化能力较程序性细胞死亡受体1过表达+神经前体细胞发育下调蛋白4敲减空载组升高，并激活PI3K/AKT通路。结果表明：程序性细胞死亡受体1能够与神经前体细胞发育下调蛋白4相互调节，影响PI3K/AKT通路活性，抑制骨髓间充质干细胞向成骨方向分化。

https://orcid.org/0009-0008-3316-573X (韩念荣) 


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程



关键词: 骨髓间充质干细胞, 程序性细胞死亡受体1(PD-1), 神经前体细胞发育下调蛋白4 (NEDD4), PI3K；AKT, 信号通路, 高糖微环境

Abstract: BACKGROUND: Programmed cell death receptor-1 (PD-1) and neural precursor cell expressed developmentally downregulated 4 (NEDD4) are involved in the regulation of osteoblast differentiation, but the specific interaction between the two and the underlying regulatory mechanism still need to be further studied. 
OBJECTIVE: To investigate the mechanism of the effect of PD-1 regulation of NEDD4 on osteogenic differentiation of rat bone marrow mesenchymal stem cells in high-glucose environment.
METHODS: (1) Immunoprecipitation-mass spectrometry was used to detect the interaction protein of PD-1. Co-immunoprecipitation was used to verify the interaction between PD-1 and NEDD4, and immunofluorescence was used to detect the localization of PD-1 and NEDD4. (2) Passage 3 rat bone marrow mesenchymal stem cells were randomly divided into normal glucose group (5.6 mmol/L), high glucose group (30 mmol/L), PD-1 knockdown empty group, PD-1 knockdown group, PD-1 overexpression empty group, and PD-1 overexpression group. Western blot assay was used to detect the protein expression of NEDD4. (3) Passage 3 rat bone marrow mesenchymal stem cells were randomly divided into normal glucose group (5.6 mmol/L), high glucose group (30 mmol/L), and NEDD4 knockdown group. qRT-PCR was used to measure the mRNA expression levels of NEDD4, zinc finger transcription factor Sp7 (OSX) and Runt related transcription factor 2 (Runx2) in each group. Alizarin Red S staining and alkaline phosphatase staining were used to evaluate their osteogenic differentiation ability. Western blot assay was used to detect the protein expression levels of Runx2, OSX, AKT, PI3K, p-PI3K, and p-AKT. (4) Subsequently, while PD-1 was overexpressed, NEDD4 knockdown treatment was performed to conduct a recovery experiment and observe changes in cell osteogenic differentiation.
RESULTS AND CONCLUSION: (1) Immunoprecipitation-mass spectrometry, co-immunoprecipitation and immunofluorescence experiments showed that NEDD4 was the interactive protein of PD-1, and PD-1 and NEDD4 were co-localized. (2) The mRNA and protein expression levels of PD-1 in NEDD4 knockdown group were decreased in high glucose group (P < 0.05). (3) NEDD4 knockdown group promoted osteogenic differentiation of rat bone marrow mesenchymal stem cells and activated PI3K/AKT pathway. (4) Osteoblast differentiation in the PD-1 overexpression + NEDD4 knockdown group was higher than that in the PD-1 overexpression + NEDD4 knockdown empty group, and the PI3K/AKT pathway was activated. It is concluded that PD-1 can regulate with NEDD4, affecting the activity of PI3K/AKT pathway and suppressing the osteogenic differentiation of bone marrow mesenchymal stem cells.


Key words: bone marrow mesenchymal stem cell, programmed cell death receptor 1 (PD-1), neural precursor cell expressed developmentally downregulated 4 (NEDD4), PI3K, AKT, signaling pathway, high-glucose microenvironment

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