Remacha Ana Rosa, Barrachina Laura, Álvarez-Arguedas Samuel, Ranera Beatriz, Romero Antonio, Vázquez Francisco José, Zaragoza Pilar, Yañez Rosa, Martín-Burriel Inmaculada, Rodellar Clementina
Laboratorio de Genética Bioquímica (LAGENBIO), Facultad de Veterinaria, Universidad de Zaragoza, 50013 Zaragoza, Spain.
Laboratorio de Genética Bioquímica (LAGENBIO), Facultad de Veterinaria, Universidad de Zaragoza, 50013 Zaragoza, Spain; Hospital Veterinario, Facultad de Veterinaria, Universidad de Zaragoza, 50013 Zaragoza, Spain.
Vet Immunol Immunopathol. 2015 Jun 15;165(3-4):107-18. doi: 10.1016/j.vetimm.2015.04.004. Epub 2015 Apr 21.
The immunomodulatory capacities of mesenchymal stem cells (MSCs) have made them the subject of increased clinical interest for tissue regeneration and repair. We have studied the immunomodulatory capacity of equine MSCs derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) in cocultures with allogeneic peripheral blood mononuclear cells (PBMCs). Different isoforms and concentrations of phytohaemaglutinin (PHA) were tested to determine the best stimulation conditions for PBMC proliferation and a proliferation assay was performed for 7 days to determine the optimal day of stimulation of PBMCs. The effect of the dose and source of MSCs was evaluated in cocultures of 10(5) PBMCs with different ratios of AT- and BM-MSCs (1:1, 1:10, 1:20 and 1:50). Proliferation rates of the PBMCs were evaluated using BrdU ELISA colorimetric assay. PHA stimulated equine PBMCs reached their peak of growth after 3 days of culture. The immunoassay showed a decrease of the PBMCs growth at high ratio cocultures (1:1 and 1:10). Equine BM-MSCs and AT-MSCs demonstrated an ability to suppress the proliferation of stimulated PBMCs. Although MSCs derived from both sources displayed immunosuppressive effects, AT-MSCs were slightly more potent than BM-MSCs. In addition, the expression of 26 genes coding for different molecules implicated in the immune response was analyzed in cocultures of BM-MSCs and PHA stimulated PBMSCs by reverse transcriptase real time quantitative PCR (RT-qPCR). An upregulation in genes associated with the production of interleukins and cytokines such as TNF-α and TGF-β1 was observed except for IFN-γ whose expression significantly decreased. The variations of interleukins and cytokine receptors showed no clear patterns. COX-1 and COX-2 showed similar expression patterns while INOs expression significantly decreased in the two cell types present in the coculture. Cyclin D2 and IDO-1 showed an increased expression and CD90, ITG-β1 and CD44 expression decreased significantly in BM-MSCs cocultured with PHA stimulated PBMCs. On the contrary, CD6 and VCAM1 expression increased in these cells. With regard to the expression of the five genes involved in antigen presentation, an upregulation was observed in both cocultured MSCs and stimulated PBMCs. This study contributes to the knowledge of the immunoregulatory properties of equine MSCs, which are notably important for the treatment of inflammation processes, such as tendinitis and osteoarthritis.
间充质干细胞（MSCs）的免疫调节能力使其成为组织再生和修复领域临床关注度不断提高的研究对象。我们研究了源自骨髓的马间充质干细胞（BM-MSCs）和脂肪组织的马间充质干细胞（AT-MSCs）与同种异体外周血单个核细胞（PBMCs）共培养时的免疫调节能力。测试了不同亚型和浓度的植物血凝素（PHA），以确定PBMC增殖的最佳刺激条件，并进行了为期7天的增殖试验，以确定PBMCs的最佳刺激时间。在10(5)个PBMCs与不同比例的AT-MSCs和BM-MSCs（1:1、1:10、1:20和1:50）的共培养中评估了MSCs的剂量和来源的影响。使用BrdU ELISA比色法评估PBMCs的增殖率。PHA刺激的马PBMCs在培养3天后达到生长峰值。免疫测定显示，在高比例共培养（1:1和1:10）时PBMCs的生长有所下降。马BM-MSCs和AT-MSCs均显示出抑制受刺激PBMCs增殖的能力。虽然源自两种来源的MSCs均表现出免疫抑制作用，但AT-MSCs的作用略强于BM-MSCs。此外，通过逆转录实时定量PCR（RT-qPCR）分析了BM-MSCs与PHA刺激的PBMSCs共培养中26种编码参与免疫反应的不同分子的基因的表达。观察到与白细胞介素和细胞因子如TNF-α和TGF-β1产生相关的基因上调，但IFN-γ的表达显著下降。白细胞介素和细胞因子受体的变化没有明显模式。COX-1和COX-2表现出相似的表达模式，而共培养中存在的两种细胞类型中INOs的表达均显著下降。在与PHA刺激的PBMCs共培养的BM-MSCs中，细胞周期蛋白D2和IDO-1表达增加，而CD90、ITG-β1和CD44表达显著下降。相反，这些细胞中CD6和VCAM1表达增加。关于参与抗原呈递的五个基因的表达，在共培养的MSCs和受刺激的PBMCs中均观察到上调。本研究有助于了解马MSCs的免疫调节特性，这对于治疗炎症过程，如肌腱炎和骨关节炎尤为重要。
