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浙江大学学报（医学版）
浙江大学学报（医学版）  2019, Vol. 48 Issue (5): 517-525    DOI: 10.3785/j.issn.1008-9292.2019.10.09
原著     
机械应力促进炎性环境中软骨修复的机制研究
姚旺祥1,2(),戴晗豪1(),桂鉴超1,*()
1. 南京医科大学附属南京医院运动关节科, 江苏 南京 210006
2. 杭州市第一人民医院骨科, 浙江 杭州 310006
Mechanical stress promotes cartilage repair in inflammatory environment
YAO Wangxiang1,2(),DAI Hanghao1(),GUI Jianchao1,*()
1. Department of Sports Medicine and Joint Surgery, the Affiliated Nanjing Hospital of Nanjing Medical University, Nanjing 210006, China
2. Department of Orthopedics, Hangzhou First People's Hospital, Hangzhou 310006, China
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摘要：

目的: 探索在炎性环境中施加机械应力对软骨修复的影响及机制。方法: 提取骨关节炎（OA）患者膝关节软骨中的软骨前体细胞（CPC），培养扩增后在海藻酸构建的支架上三维培养，在IL-1β塑造的炎症环境中施加周期性静水压（IHP），蛋白免疫印迹法检测MAPK信号通路的变化，CCK-8法检测细胞增殖，实时荧光定量PCR检测基质金属蛋白酶13（MMP-13）和解聚素与金属蛋白酶5（ADAMTS-5）基因表达的变化。切断大鼠的前交叉韧带构建膝关节OA模型，通过外固定支架牵开关节腔构建合适的机械应力，观察关节牵引能否促进软骨的修复及其机制。结果: 0.01 ng/mL IL-1β可以抑制CPC增殖，施加IHP后可以减少MAPK信号通路蛋白的表达，减少MMP-13、ADAMTS-5基因表达，促进炎性环境下的CPC增殖。切断前交叉韧带4周后，膝关节OA模型成功构建。OA大鼠经关节牵引后软骨的Mankin评分更低（P < 0.01），软骨修复较对照组更佳。软骨标本的免疫组织化学染色发现关节牵引可减少P-p38、MMP-13及X型胶原的表达，减少细胞凋亡，促进OA软骨的修复。结论: 机械应力可以通过抑制MAPK信号通路，促进CPC增殖，减少基质降解酶表达，促进OA软骨修复。
关键词： 骨关节炎/病理学;  压力;  软骨细胞;  白细胞介素1β;  基质金属蛋白酶13;  丝裂原激活蛋白激酶类;  细胞, 培养的;  疾病模型, 动物    
Abstract:

Objective: To investigate the effect and mechanism of mechanical stress on cartilage repair in inflammatory environment. Methods: The chondrogenic progenitor cells (CPCs) were isolated from the knee joint cartilage of patients with osteoarthritis (OA) undergoing total knee arthroplasty. The CPCs were cultured and expanded in a 3-D scaffold constructed with alginate. Intermittent hydrostatic pressure (IHP) was applied in a inflammatory environment induced by IL-1β, and Western blot was used to detect the expression of MAPK signaling pathway proteins. Cell proliferation was detected by CCK-8 method, and the expression of related genes like matrix metallo-proteinases 13 (MMP-13) and a disintegrins and metalloproteinase with thrombospondin motif 5 (ADAMTS-5) was detected by real-time RT-PCR. The anterior cruciate ligament of the rats was cut to construct the knee joint OA model, and the appropriate mechanical stress was constructed with external fixation to distract the knee joint in order to observe the repair of the cartilage and to explore its mechanism. Results: Adding 0.01 ng/ml IL-1β in cell culture inhibited the proliferation of CPCs. After IHP application, the expression of MAPK pathway protein was decreased, the mRNA expression of MMP-13 and ADAMTS-5 was reduced. The inhibition of IL-1β on CPCs was counteracted by IHP. Four weeks after the anterior cruciate ligament resected, the articular cartilage degeneration was observed in rats. The Mankin score in the OA treatment (joint distraction) group was lower, and the cartilage repair was better than that of the control group (P < 0.01). Animal experiments found that the suitable mechanical stress reduced the expression of P-p38, MMP-13 and COLL-X, inhibited cartilage cells apoptosis and promoted the repair of OA cartilage. Conclusion: Mechanical stress can promote the proliferation of CPCs, reduce the expression of matrix degrading enzymes, and promote the repair of OA cartilage by inhibiting MAPK signaling pathway.
Key words: Osteoarthritis/pathology    Pressure    Chondrocytes    Interleukin-1beta    Matrix metalloproteinase 13    Mitogen-activated protein kinases    Cells, cultured    Disease models, animal
收稿日期: 2019-07-03 出版日期: 2020-01-04
:  R684.3  
基金资助: 国家自然科学基金(81672210)
通讯作者: 桂鉴超     E-mail: yaowx04@163.com;a674503005@163.com;gui1997@126.com
作者简介: 姚旺祥(1981—), 男, 硕士, 副主任医师, 主要从事骨关节炎及软骨修复的机制研究; E-mail: yaowx04@163.com; https://orcid.org/0000-0003-2962-5330|戴晗豪(1994—), 男, 硕士研究生, 主要从事骨关节炎及软骨修复的机制研究; E-mail: a674503005@163.com; https://orcid.org/0000-0002-8197-3183
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引用本文:

姚旺祥,戴晗豪,桂鉴超. 机械应力促进炎性环境中软骨修复的机制研究[J]. 浙江大学学报（医学版）, 2019, 48(5): 517-525.

YAO Wangxiang,DAI Hanghao,GUI Jianchao. Mechanical stress promotes cartilage repair in inflammatory environment. J Zhejiang Univ (Med Sci), 2019, 48(5): 517-525.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2019.10.09    或    http://www.zjujournals.com/med/CN/Y2019/V48/I5/517

基因名称序列(5′→3′)
MMP-13正向：5-CCAGACTTCACGATGGCATTG-3
反向：5-GGCATCTCCTCCATAATTTGGC-3
ADAMTS-5正向：5-TGACAAGTGCGGAGTATG-3
反向：5-AGGCAGTGAATCTAGTCTGG-3
表 1  实时荧光定量PCR引物序列
图 1  软骨前体细胞的细胞生长形态
图 2  IL-1β及施加周期性静水压对软骨前体细胞增殖的影响
图 3  预培养一周的细胞-海藻酸微球施加机械应力可抑制IL-1β对MAPK信号通路的激活
图 4  IL-1β环境下施加机械应力可减少软骨前体细胞中MMP-13、ADAMTS-5 mRNA表达
图 5  治疗组和对照组膝关节股骨髁软骨的大体观、X线摄片和组织病理学检查结果
图 6  软骨标本的TUNEL染色观察软骨细胞凋亡情况
图 7  对照组与治疗组软骨组织中P-p38、MMP-13与COLL-X表达
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