中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (1): 8-15.doi: 10.12307/2024.734

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

温肾通络止痛方对小鼠H型骨微血管内皮细胞 / 骨髓间充质干细胞共培养体系的影响

周士杰 1,李沐哲 1,员 浬 1,张天驰 1,牛园园 1,朱弈桦 1,周勤峰 1,郭 杨 1,2,马 勇 1,2,3,王礼宁 1,3   

  1. 南京中医药大学,1 骨伤修复与重建新技术实验室,3 中医学院 · 中西医结合学院,江苏省南京市 210023;2 南京中医药大学无锡附属医院,江苏 省中医退行性骨关节病临床医学创新中心,江苏省无锡市 214071
  • 收稿日期:2023-10-17 接受日期:2023-12-11 出版日期:2025-01-08 发布日期:2024-05-18
  • 通讯作者: 王礼宁,讲师,南京中医药大学,骨伤修复与重建新技术实验室,中医学院 · 中西医结合学院,江苏省南京市 210023
  • 作者简介:周士杰,男,1997 年生,江苏省南京市人,汉族,南京中医药大学在读博士,主要从事中西医结合治疗骨伤科疾病的研究。
  • 基金资助:
    国家自然科学基金面上项目 (82074458),项目负责人:郭杨;江苏省自然科学基金面上项目 (BK20221351),项目负责人:郭杨;江苏 省自然科学基金青年项目 (BK20220470),项目负责人:王礼宁;江苏省高等学校自然科学研究面上项目 (22KJB360012),项目负责人:王礼宁;国 家中医药管理局高水平中医药重点学科建设项目 ( 国中医药人教函〔2023〕85 号 ),项目负责人:马勇;江苏省中医退行性骨关节病临床医学创新 中心资助项目 ( 苏中医科教〔2021〕4 号 ),项目负责人:马勇

Effect of Wenshen Tongluo Zhitong formula on mouse H-type bone microvascular endothelial cell/bone marrow mesenchymal stem cell co-culture system

Zhou Shijie1, Li Muzhe1, Yun Li1, Zhang Tianchi1, Niu Yuanyuan1, Zhu Yihua1, Zhou Qinfeng1, Guo Yang1, 2, Ma Yong1, 2, 3, Wang Lining1, 3   

  1. 1New Technology Laboratory of Bone Injury Repair and Reconstruction, Nanjing University of Chinese Medicine, Nanjing 210023, Jiangsu Province, China; 2 Wuxi Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Clinical Medical Innovation Center for Degenerative Osteoarthropathy of Traditional Chinese Medicine, Wuxi 214071, Jiangsu Province, China; 3 School of Chinese Medicine & School of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, Jiangsu Province, China
  • Received:2023-10-17 Accepted:2023-12-11 Online:2025-01-08 Published:2024-05-18
  • Contact: Wang Lining, Lecturer, New Technology Laboratory of Bone Injury Repair and Reconstruction, Nanjing University of Chinese Medicine, Nanjing 210023, Jiangsu Province, China; School of Chinese Medicine & School of Integrated Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, Jiangsu Province, China
  • About author:Zhou Shijie, Doctoral candidate, New Technology Laboratory of Bone Injury Repair and Reconstruction, Nanjing University of Chinese Medicine, Nanjing 210023, Jiangsu Province, China
  • Supported by:
    National Natural Science Foundation of China, No. 82074458 (to GY); Natural Science Foundation Project of Jiangsu Province, No. BK20221351 (to GY); Youth Project of Natural Science Foundation of Jiangsu Province, No. BK20220470 (to WLN); Jiangsu Provincial Colleges and Universities Natural Science Research Project, No. 22KJB360012 (to WLN); NATCM's Project of High-level Construction of Key TCM Disciplines (NATCM’s Human Education Letter[2023]No. 85) (to MY); Funded Project of Jiangsu Provincial Clinical Medicine Innovation Center for Degenerative Osteoarthrosis of Traditional Chinese Medicine (JSTCM’s Science and Education[2021]No. 4) (to MY) 

摘要:

文题释义: 

温肾通络止痛方:是以“络病”学说为理论基础、“荣骨通络”为核心理念、多年临床治疗经验为积累所构建的临床经验方,可有效缓解 骨质疏松症患者的临床症状。 

低氧环境:是指组织中氧气体积分数低于5%。低氧环境是许多组织中正常的生理状态,如骨髓、肠黏膜、胎盘、视网膜等,并可以激活 低氧反应,维持正常的生理稳态。 


背景:骨骼依赖血管与骨细胞之间的密切连接来维持完整性,骨骼处在生理低氧环境中,因此在低氧环境下研究血管生成与骨生成具有重 要意义。 

目的:探究在低氧环境下温肾通络止痛方对H型骨微血管内皮细胞/骨髓间充质干细胞共培养体系的影响及相关机制。 

方法:运用酶消化法及流式分选技术,分选小鼠H型血管特异型骨微血管内皮细胞;应用骨髓贴壁法分离获取小鼠骨髓间充质干细胞;采 用Transwell小室以及缺氧培养工作站建立两种细胞低氧共培养体系,使用50,100 μg/mL质量浓度温肾通络止痛方冻干粉进行干预;通过 划痕迁移实验与管形成实验评估共培养体系内H型骨微血管内皮细胞的成血管功能;通过碱性磷酸酶染色与茜素红染色评估共培养体系内 骨髓间充质干细胞的成骨分化能力;使用Western Blotting与q-PCR检测共培养体系内H型骨微血管内皮细胞中PDGF/PI3K/AKT信号轴相关分 子的蛋白表达及mRNA表达。 

结果与结论:①与常氧组相比,低氧组H型骨微血管内皮细胞划痕迁移率及新生血管长度均显著升高(P < 0.05),骨髓间充质干细胞成骨分 化能力增强(P < 0.05);PDGF/PI3K/AKT信号轴相关分子的蛋白、mRNA表达升高(P < 0.05);②与低氧组相比,经不同质量浓度温肾通络止痛 方干预后H型骨微血管内皮细胞划痕迁移率及新生血管长度进一步提高(P < 0.05),骨髓间充质干细胞成骨分化能力更强(P < 0.05),PDGF/ PI3K/AKT信号轴相关分子的蛋白、mRNA表达也进一步升高(P < 0.05)。结果表明:低氧条件下H型血管生成及骨生成可能与PDGF/PI3K/AKT 信号轴有关,而温肾通络止痛方可能通过激活PDGF/PI3K/AKT信号轴促进“H型血管生成-骨生成”作用从而防治骨质疏松症。

https://orcid.org/0000-0001-7367-2244 ( 周士杰 );https://orcid.org/0000-0001-6287-5724 ( 王礼宁 )


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: 温肾通络止痛方, H型骨微血管内皮细胞, 骨髓间充质干细胞, 低氧, 血管生成, 骨生成

Abstract:

BACKGROUND: Bone relies on the close connection between blood vessels and bone cells to maintain its integrity. Bones are in a physiologically hypoxic environment. Therefore, the study of angiogenesis and osteogenesis in hypoxic environment is closer to the microenvironment in vivo. 

OBJECTIVE: To explore the influence of Wenshen Tongluo Zhitong (WSTLZT) formula on H-type bone microvascular endothelial cell/bone marrow mesenchymal stem cell co-culture system in hypoxia environment and its related mechanism. 

METHODS: Enzyme digestion method and flow sorting technique were used to isolate and identify H-type bone microvascular endothelial cells. Mouse bone marrow mesenchymal stem cells were isolated and obtained by bone marrow adhesion method. H-type bone microvascular endothelial cell/bone marrow mesenchymal stem cell hypoxic co-culture system was established using Transwell chamber and anoxic culture workstation. WSTLZT formula powder was used to intervene in each group at a mass concentration of 50 and 100 μg/mL. The angiogenic function of H-type bone microvascular endothelial cells in the co-culture system was evaluated by scratch migration test and tube formation test. The osteogenic differentiation ability of bone marrow mesenchymal stem cells in the cocultured system was evaluated by alkaline phosphatase staining and alizarin red staining. The protein and mRNA expression changes of PDGF/PI3K/AKT signal axis related molecules in H-type bone microvascular endothelial cells in the co-cultured system were detected by Western Blotting and q-PCR, respectively. 

RESULTS AND CONCLUSION: (1) Compared with the normal oxygen group, the scratch mobility and new blood vessel length of H-type bone microvascular endothelial cells were significantly higher (P < 0.05); the osteogenic differentiation capacity of bone marrow mesenchymal stem cells was higher (P < 0.05); the expression of PDGF/PI3K/AKT axis-related molecular protein and mRNA increased (P < 0.05) in the hypoxia group. (2) Compared with the hypoxia group, scratch mobility and new blood vessel length were significantly increased in the H-type bone microvascular endothelial cells (P < 0.05); bone marrow mesenchymal stem cells had stronger osteogenic function (P < 0.05); the expression of PDGF/PI3K/AKT axis-related molecular proteins and mRNA further increased (P < 0.05) after treatment with different dose concentrations of WSTLZT formula. These findings conclude that H-type angiogenesis and osteogenesis under hypoxia may be related to the PDGF/PI3K/AKT signaling axis, and WSTLZT formula may promote H-type vasculo-dependent bone formation by activating the PDGF/PI3K/AKT signaling axis, thereby preventing and treating osteoporosis.

Key words: Wenshen Tongluo Zhitong formula, H-type bone microvascular endothelial cell, bone marrow mesenchymal stem cell, hypoxia, angiogenesis, osteogenesis

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