中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (6): 866-871.doi: 10.12307/2023.250

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

人血管周围干细胞成骨分化及Wnt/β-catenin信号通路的调控

袁  维1，刘静东1，徐广辉1，康  健1，李富平1，王英杰1，支中正1，李冠武2   

  1. 1同济大学附属上海市第四人民医院脊柱外科，上海市   200434；2上海中医药大学附属岳阳中西医结合医院医学影像科，上海市   200437
  • 收稿日期:2022-02-08 接受日期:2022-04-23 出版日期:2023-02-28 发布日期:2022-08-11
  • 通讯作者: 刘静东，主任医师，同济大学附属上海市第四人民医院脊柱外科，上海市 200434 李冠武，博士，副主任医师，上海中医药大学附属岳阳中西医结合医院医学影像科，上海市 200437
  • 作者简介:袁维，男，1975年生，辽宁省沈阳市人，汉族，2004年中国医科大学毕业，博士，主要从事骨组织工程及椎间盘退变研究。
  • 基金资助:
    上海市卫生健康委员会科研课题面上项目(202040317)，项目负责人：袁维；上海市第四人民医院学科助推计划项目(SY-XKZT-2021-1009)，项目负责人：袁维；上海市虹口区重点扶持专科资助项目(HKZK2020A07)，项目负责人：徐广辉

Osteogenic differentiation of human perivascular stem cells and its regulation based on Wnt/beta-catenin signaling pathway

Yuan Wei1, Liu Jingdong1, Xu Guanghui1, Kang Jian1, Li Fuping1, Wang Yingjie1, Zhi Zhongzheng1, Li Guanwu2   

  1. 1Department of Spine Surgery, Shanghai Fourth People’s Hospital, Tongji University, Shanghai 200434, China; 2Department of Radiology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200437, China
  • Received:2022-02-08 Accepted:2022-04-23 Online:2023-02-28 Published:2022-08-11
  • Contact: Liu Jingdong, Chief physician, Department of Spine Surgery, Shanghai Fourth People’s Hospital, Tongji University, Shanghai 200434, China Li Guanwu, MD, Associate chief physician, Department of Radiology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 200437, China
  • About author:Yuan Wei, MD, Department of Spine Surgery, Shanghai Fourth People’s Hospital, Tongji University, Shanghai 200434, China
  • Supported by:
    the General Program of Shanghai Municipal Health Commission, No. 202040317 (to YW); Academic Subject Boosting Plan in the Shanghai Fourth People’s Hospital, No. SY-XKZT-2021-1009 (to YW); Key Support to Specialty-Funded Project of Shanghai Hongkou District, No. HKZK2020A07 (to XGH)

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摘要：

文题释义：
血管周围干细胞：脂肪组织的血管中可提取一种干细胞，包括周细胞和外膜细胞两类，合并称为血管周围干细胞。血管周围干细胞与间充质干细胞存在相似性，可作为组织工程的种子细胞。
Wnt/β-catenin信号通路：是Wnt通路的一种经典途径，与干细胞的成骨分化密切相关。当存在Wnt信号刺激时，GSK-3β活性受到抑制，β-catenin在胞质内大量积聚并进入细胞核与TCF/LEF家族的转录因子结合，从而启动下游成骨靶基因的表达。

背景：血管周围干细胞来源于成体脂肪组织，获取容易且干细胞比较均质，具有较强的增殖和多向分化潜能。
目的：通过体外、体内实验探讨人血管周围干细胞的成骨分化能力，以及Wnt/β-catenin信号通路的调控作用。
方法：抽脂来源的人脂肪组织，经荧光激活细胞分选法提取血管周围干细胞，第1代血管周围干细胞进行成骨诱导分化，分为空白对照组、成骨诱导组及Wnt信号通路抑制组，成骨诱导第5天行碱性磷酸酶染色，第10天行茜素红染色，第7天通过Western blot检测Runt相关转录因子2蛋白表达。制备血管周围干细胞复合纳米羟基磷灰石/聚乳酸-羟基乙酸共聚物支架材料，通过无胸腺小鼠胫骨单皮质缺损模型，研究血管周围干细胞在体内的骨修复效果。
结果与结论：①每100 mL脂肪中含有基质血管成分细胞为(1.82±0.32)×107，活细胞占(82.72±5.37)%，经荧光激活细胞分选法分选，血管外膜细胞(CD34+、CD45-、CD146-)比例为(17.66±1.05)%、血管外周细胞(CD146+、CD45-、CD34-)比例为(7.18±0.52)%，血管周围干细胞体外扩增迅速，10代之内保持较强的增殖能力；②细胞实验显示血管周围干细胞具有成骨分化能力，抑制Wnt信号通路后Runt相关转录因子2表达明显减少，血管周围干细胞成骨分化受到抑制，动物实验进一步证实血管周围干细胞复合支架材料的成骨效果；③结果表明：Wnt/β-catenin信号通路在血管周围干细胞成骨分化中发挥重要作用，血管周围干细胞为骨修复提供一种新的治疗选择。

https://orcid.org/0000-0003-3766-5383 (袁维) 

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: 人脂肪组织, 血管周围干细胞, 细胞分选法, 成骨分化, 信号通路

Abstract: BACKGROUND: Perivascular stem cells are derived from adult adipose tissue, which can be easily purified. Perivascular stem cells represent a comparatively homogenous population and have the ability of strong proliferation and multidirectional differentiation. 
OBJECTIVE: To explore the osteogenic differentiation ability of human perivascular stem cells and the regulation by Wnt/β-catenin signaling pathway by in vitro and in vivo experiments.
METHODS:  Perivascular stem cells were extracted from human lipoaspirate via fluorescence-activated cell sorting. Osteogenic differentiation of passage 1 perivascular stem cells was observed. Cells were randomly divided into blank control group, osteogenic differentiation group and Wnt/β-catenin signaling inhibition group. Alkaline phosphatase staining was performed on day 5 of osteogenic induction. Alizarin red staining was performed on day 10. On day 7, the protein expression of Runt related transcription factor 2 was detected by western blot assay. Perivascular stem cells + nano-hydroxyapatite/poly lactic-co-glycolic acid scaffold was prepared. Bone repair effect of perivascular stem cells in vivo was evaluated by using the model of tibia monolayer cortical bone defect in athymic mice. 
RESULTS AND CONCLUSION: (1) There were about (1.82±0.32)×107 stromal vascular fraction cells per 100 mL of fat tissue, and of which the living cells accounted for (82.72±5.37)%. Through fluorescence-activated cell sorting, the proportion of adventitial cells (CD34+, CD45-, CD146-) was (17.66±1.05)%; and the proportion of pericytes (CD146+, CD45-, CD34-) was (7.18±0.52)%. Perivascular stem cells proliferated rapidly and still maintained strong proliferative ability within 10 generations. (2) In vitro studies had confirmed that perivascular stem cells had the ability of osteogenic differentiation. In addition, the expression of Runt-related transcription factor 2 (RUNX2) was significantly reduced and osteogenic differentiation of perivascular stem cells was attenuated by inhibition of Wnt signaling pathway. Animal experiments further confirmed the osteogenic effect of perivascular stem cell composite scaffolds. (3) These results indicate that Wnt/β-catenin signaling pathway plays an important role in the osteogenic differentiation of perivascular stem cells, which provides a new therapeutic option for bone repair. 

Key words: human fat tissue, perivascular stem cell, fluorescence activated cell sorting, osteogenic differentiation, signaling pathway

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