| 巨噬细胞过表达Wnt5a对其炎性反应及共培养体系中MSCs成软骨分化的影响研究 |
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Received:August 21, 2019
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| 期刊信息：《中国骨伤》2019年32卷，第11期，第1026-1033页 |
| DOI：10.3969/j.issn.1003-0034.2019.11.010 |
| 基金项目:国家自然科学基金资助项目（编号：81672183）；浙江省医药卫生一般研究计划项目（编号：2018KY204）；深圳市科技研发计划资助项目号（编号：JCYJ20170307111755218）；北京市自然科学基金资助项目（编号：7182172）；北京大学人民医院院内发展基金资助项目（编号：RDY2016-15，RDY2018-04） |
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目的：通过构建和比较经Wnt5a基因修饰的巨噬细胞分别与骨髓干细胞（MSCs）2D单层贴壁与骨髓液跨小室共培养模型，旨在探索Wnt5a信号通过调控巨噬细胞炎症反应对MSCs成软骨分化产生的影响。
方法：自2015年9月至2018年12月收集6例重度膝关节骨关节炎拟行全膝关节置换术者的巨噬细胞、MSCs和骨髓液，其中男2例，女4例；年龄58~71岁。膝关节囊滑膜组织常规消化取得单细胞，经反复贴壁并行Ficoll液密度梯度离心和抗CD14抗体流式细胞术测定巨噬细胞纯度。将上述经鉴定的巨噬细胞置于IFN-γ联合TNF-α刺激48 h后，再使用重组腺相关病毒（rAAV）-lacZ或Wnt5a转染24 h，分别与MSCs 2D单层贴壁或骨髓液构建跨小室共培养模型，采用HE染色、软骨特殊染色、X-gal染色、抗Wnt5a、抗Ⅱ和X型胶原免疫组化染色对细胞形态与增殖能力、病毒转染效率和成软骨分化能力等进行检测。
结果：抗CD68免疫组化显示骨关节炎患者滑膜组织巨噬细胞明显增多。抗CD14流式细胞术证实经分离的滑膜巨噬细胞纯度为90.31%，IFN-γ联合TNF-α刺激后发现上清液中单核细胞趋化素蛋白-1（MCP-1）水平明显升高，提示巨噬细胞被激活；rAAV-lacZ转染3 d后，X-gal染色发现其能有效转染巨噬细胞，效率高达97.50%，且至少持续21 d；rAAV-Wnt5a转染巨噬细胞后通过刺激其Wnt5a的表达，而增加两种模型中的Wnt5a表达，抑制MCP-1的分泌，两种模型中Wnt5a组MCP-1表达量分别为14.76和61.51 pg/ml；此外，rAAV-Wnt5a转染后能促进细胞增殖及成软骨分化、软骨基质合成。
结论：在巨噬细胞与MSCs 2D单层贴壁或骨髓液跨小室共培养条件下，rAAV介导的Wnt5a过表达能通过影响巨噬细胞炎症反应而促进软骨稳态的维持以及MSCs成软骨分化，巨噬细胞可能通过Wnt5a信号影响软骨稳态与MSCs成软骨分化。 |
| [关键词]:巨噬细胞 Wnt5a蛋白 软骨细胞 干细胞 骨髓 |
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| Effects of macrophage with Wnt5a over-expression on inflammation and chondrogenic differentiation in co-culture system |
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Abstract:
Objective: To construct and compare macrophage by gene modification of Wnt5a which co-cultured by human bone marrow-derived mesenchymal stem cells (MSCs) with two dimension (2D) and bone marrow aspirates in vitr transwell,in order to investigate the effect of Wnt5a signaling on cartilage homeostasis through regulation of macrophage pro-inflammatory responses.
Methods: Macrophages,MSCs and bone marrow aspirates specimens were extracted from 6 patients with severe knee deformities undergoing total knee arthroplasty(2 males,4 females,aged from 58 to 71 years old) from September 2015 to December 2018. The synovial tissues of knee joints were exposed to typeⅡcollagenase and obtained single cell suspensions,and the purity of macrophages was determined by Ficoll gradient centrifugation and anti-CD14 antibody flow cytometry. The macrophages were transduced with IFN-γ combined with TNF-α for 48 h,and rAAV-lacZ or Wnt5a transfected for 24 h,then co-cultured model by human bone marrow-derived mesenchymal stem cells (MSCs) with two dimension (2D) and bone marrow aspirates in vitr transwell. HE staining,toluidine blue staining,X-gal staining and anti-Wnt5a,anti-Ⅱ,X collagen immunohistochemical staining and enzyme-linked immunosorbent assay were applied to detect cell morphology and proliferation (cellularity),viral transfection efficiency respectively.
Results: Results of anti-CD68 immunohistochemistry showed macrophage of patients with osteoarthritis increased obviously. Anti-CD 14 flow cytometry confirmed that percentage of isolated synovial macrophages was 90.31%. The level of monocyte chemotactic protein-1 in supernatants was significantly increased after stimulation with IFN-γ and TNF-α,indicating that the macrophages were activated and at proinflammatory condition. After transduction with rAAV-lacZ for 3 days,X-gal staining indicated that lacZ gene transfer could efficiently transduce the activated macrophages with the efficiency over 97.50% for at least 21 days. After? transfection macrophages by rAAV-Wnt5a stimulated expression of Wnt5a,and enhanced expression of Wnt5a between two models and inhibited the secretion of MCP-1. The expression of MCP-1 in Wnt5a group was 14.76 and 61.51 pg/ml respectively. In addition,rAAV-Wnt5a gene transfer could promote cell proliferation,chondrogenic differentiation and cartilage matrix synthesis.
Conclusion: Under the condition of macrophage and MSCs 2D monolayer or aspirates transwell co-culture,rAAV-mediated over-expression of Wnt5a could promote maintenance of cartilage homeostasis and chondrogenic differentiation of MSCs via macrophage inflammatory response,macrophages may affect cartilage homeostasis and MSCs chondrogenesis through Wnt5a signaling pathway. |
| KEYWORDS:Macrophages Wnt-5a protein Chondrocytes Stem cells Bone marrow |
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| 引用本文，请按以下格式著录参考文献： |
| 中文格式: | 桂先革,曾晖,陶可.巨噬细胞过表达Wnt5a对其炎性反应及共培养体系中MSCs成软骨分化的影响研究[J].中国骨伤,2019,32(11):1026~1033 |
| 英文格式: | GUI Xian-ge,ZENG Hui,TAO Ke.Effects of macrophage with Wnt5a over-expression on inflammation and chondrogenic differentiation in co-culture system[J].zhongguo gu shang / China J Orthop Trauma ,2019,32(11):1026~1033 |
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