肌腱去应力退化机制的生物信息学分析

doi:10.3969/j.issn.1674-8115.2023.05.006

上海交通大学学报（医学版） ›› 2023, Vol. 43 ›› Issue (5): 560-570.doi: 10.3969/j.issn.1674-8115.2023.05.006

• 论著 · 基础研究 • 上一篇

肌腱去应力退化机制的生物信息学分析

李清林¹(), 王文波²(), 刘伟²()

^1.潍坊医学院整形外科研究所，潍坊 261042
^2.上海交通大学医学院附属第九人民医院整复外科，上海市组织工程研究重点实验室，上海 200011

收稿日期:2023-01-03 接受日期:2023-04-28 出版日期:2023-05-28 发布日期:2023-07-11
通讯作者: 王文波,刘伟 E-mail:qinglinli346@163.com;wangwenbo0903@126.com;liuwei_md@126.com
作者简介:李清林（1989—），男，硕士生；电子信箱：qinglinli346@163.com。
基金资助:
国家自然科学基金(31870967);国家重点研发计划(2018YFC1105800)

Bioinformatics analysis of pathological mechanism of degenerated tendon via stress deprivation

LI Qinglin¹(), WANG Wenbo²(), LIU Wei²()

^1.Plastic Surgery Research Institute, Weifang Medical University, Weifang 261042, China
^2.Department of Plastic and Reconstructive Surgery, Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine; Shanghai Tissue Engineering Key Laboratory, Shanghai 200011, China

Received:2023-01-03 Accepted:2023-04-28 Online:2023-05-28 Published:2023-07-11
Contact: WANG Wenbo,LIU Wei E-mail:qinglinli346@163.com;wangwenbo0903@126.com;liuwei_md@126.com
Supported by:
National Natural Science Foundation of China(31870967);National Key R&D Program of China(2018YFC1105800)

摘要/Abstract

摘要：

目的·建立新生大鼠发育肌腱去应力模型，通过生物信息学分析可能的分子机制。方法·选择60只出生10 d的SD大鼠，切断左侧跟腱作为实验组（去应力组），钳夹损伤右侧跟腱作为对照组（力刺激组）。手术处理后第10日和第20日收集肌腱组织样本，大体观察肌腱发育情况；苏木精-伊红（H-E）染色观察肌腱组织学结构变化.；透射电子显微镜观察肌腱胶原超微结构的变化；免疫组织化学检测肌腱组织中CD31的表达。运用蛋白质谱分析技术获得2个时间点的组间差异表达蛋白，并对差异蛋白行GO分析、KEGG富集分析、蛋白互作（protein-protein interaction，PPI）网络分析。结果·去应力组肌腱呈现组织膨大及充血的大体观，以及肌腱组织结构紊乱、胶原纤维不成熟的病理改变。透射电子显微镜下观察到去应力组肌腱胶原纤维发育和成熟障碍；术后第20日，去应力组肌腱胶原纤维直径显著小于力刺激组（P=0.001）。免疫组织化学染色结果显示肌腱呈血管化改变。蛋白质谱分析结果显示：术后第10日鉴定出差异表达蛋白1 865种，其中1 835种蛋白上调；术后第20日鉴定出差异表达蛋白965种，其中837种蛋白上调。GO分析结果显示，上调的差异表达蛋白主要参与细胞内蛋白运输、蛋白质稳定性、mRNA剪接、蛋白质折叠、蛋白质导入细胞核等生物过程。KEGG分析结果显示，血管内皮生长因子（VEGF）信号通路、哺乳动物雷帕霉素靶蛋白（mTOR）信号通路、吞噬作用等信号通路增强。PPI分析结果显示，Akt1、Hspa4、Hspa5、Eef2、ACTC1、RhoA等多种蛋白表达上调。结论·去应力可激活细胞内多条信号通路，引起组织血管化、胶原发育异常等，造成肌腱退化性病理改变。

关键词: 去应力刺激, 肌腱退化, 胶原, 蛋白组学, 生物信息学, 信号通路

Abstract:

Objective ·To explore relevant molecular mechanisms of the stress deprivation model of newborn rats by using bioinformatics analysis. Methods ·A total of 60 SD rats (10 d post-natal) were enrolled in the study. Those the left Achilles tendon of which was severed were chosen as the experimental group (stress deprivation group), and the right Achilles tendon of which was injured by clamping were chosen as the control group (stress group). On the 10th and 20th day after treatment, tissue samples were collected for gross observation of the tendon development, histological staining of the tendon structure, transmission electron microscope observation of the tendon ultrastructure and immunohistochemical analysis of CD31 expression. Differentially expressed proteins between the two groups at two time points were obtained by using protein mass spectrometry, and GO and KEGG enrichment analysis as well as protein-protein interaction (PPI) network anlysis were performed on differential proteins. Results ·The stress-deprived tendon showed tissue enlargement and congestion, disorganized tendon tissue structure and immature collagen fibers. Transmission electron microscopy showed that the development and maturation of collagen fibrils were significantly impaired in the experimental group, and the diameter of collagen fibrils of the severed tendon became thinner on the 20th day after treatment (P=0.001). Immunohistochemistry showed that the severed tendon was relatively vascularized. The results of protein mass spectrometry analysis showed there were 1 865 and 965 differentially expressed proteins on the 10th and 20th day after treatment, including 1 835 and 837 upregulated proteins respectively. GO analysis showed that the upregulated proteins were involved in biological processes such as intracellular protein transport, protein stabilization, mRNA splicing via spliceosome, protein folding and protein import into nucleus. KEGG analysis indentified enhancement of vascular endothelial growth factor (VEGF) signal pathway, mammalian target of rapamycin (mTOR) signal pathway, endocytosis and other signal pathways in the experimental group. PPI network analysis showed various upregulated proteins including Akt1, Hspa4, Hspa5, Eef2, ACTC1 and RhoA. Conclusion ·Stress deprivation can activate multiple signal pathways in tendon cells and lead to tissue vascularization, abnormal collagen development, etc., resulting in degenerative pathological changes of tendons.

Key words: stress deprivation, tendon degeneration, collagen, proteomics, bioinformatics, signal pathway

中图分类号:

R686

李清林, 王文波, 刘伟. 肌腱去应力退化机制的生物信息学分析[J]. 上海交通大学学报（医学版）, 2023, 43(5): 560-570.

LI Qinglin, WANG Wenbo, LIU Wei. Bioinformatics analysis of pathological mechanism of degenerated tendon via stress deprivation[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(5): 560-570.

图/表 7

图1 应力和去应力刺激的肌腱大体观和H-E染色观察Note： A. Macroscopic examination of tendon. B.H-E staining of tendon (Scale bars=200 μm).

Fig 1 Gross view and H-E staining of tendon with and without mechanical loading

图2 去应力组肌腱的组织血管化Note： Immunohistochemical staining images of CD31 (Scale bars=100 μm). The arrows showed CD31-stained vessels.

Fig 2 Tissue vascularization in stress-deprived tendon

图3 实验组和对照组肌腱胶原超微结构和胶原纤维直径比较Note： A. TEM of fibrils in tendon tissues (Scale bars=50 nm). B. Quantitative analysis of collagen fibril diameter.

Fig 3 Ultrastructural comparison of tendon with and without mechanical loading and quantitative analysis of fibril diameters

图4 去应力肌腱和应力刺激肌腱之间差异表达蛋白火山图和聚类热图Note： A/B. Differentially expressed protein volcano maps on the 10th (A) and 20th (B) day. C/D. Differential expression cluster heat maps on the 10th (C) and 20th (D) day.

Fig 4 Volcanic diagram and cluster thermogram of differentially expressed proteins between tendons with and without mechanical loading

图5 去应力肌腱和力刺激肌腱之间差异蛋白GO分析Note： A and B represent the differentially expressed protein GO analysis between stress deprivation and stress-stimulated tendons on the 10th and 20th day after surgery, respectively.

Fig 5 GO analysis of differentially expressed proteins between tendons with and without mechanical loading

图6 去应力肌腱和应力刺激肌腱之间差异蛋白的KEGG分析Note： A and B represent KEGG analysis of differentially expressed protein between stress deprivation and stress-stimulated tendons on the 10th and 20th day after surgery, respectively.

Fig 6 KEGG analysis of differentially expressed proteins between tendon with and without mechanical loading

图7 去应力肌腱和力刺激肌腱之间差异蛋白的PPI分析

Fig 7 PPI analysis of differential proteins between tendon with and without mechanical loading

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肌腱去应力退化机制的生物信息学分析

Bioinformatics analysis of pathological mechanism of degenerated tendon via stress deprivation

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