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首页> 美国卫生研究院文献>Tissue Engineering. Part A >Conditioned Medium Derived from Notochordal Cell-Rich Nucleus Pulposus Tissue Stimulates Matrix Production by Canine Nucleus Pulposus Cells and Bone Marrow-Derived Stromal Cells
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Conditioned Medium Derived from Notochordal Cell-Rich Nucleus Pulposus Tissue Stimulates Matrix Production by Canine Nucleus Pulposus Cells and Bone Marrow-Derived Stromal Cells

机译：来源于脊索细胞丰富的髓核组织的条件培养基刺激了犬髓核细胞和骨髓衍生的基质细胞的基质产生

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>Objectives: Conditioned medium derived from notochordal cell-rich nucleus pulposus tissue (NCCM) was previously shown to have a stimulatory effect on bone marrow stromal cells (BMSCs) and nucleus pulposus cells (NPCs) individually, in mixed species in vitro cell models. The objective of the current study was to assess the stimulatory effect of NCCM on NPCs in a homologous canine in vitro model and to investigate whether combined stimulation with NCCM and addition of BMSCs provides a synergistic stimulatory effect.>Methods: BMSCs and NPCs were harvested from chondrodystrophic dogs with confirmed early intervertebral disc (IVD) degeneration. NCCM was produced from NP tissue of nonchondrodystrophic dogs with healthy IVDs. BMSCs or NPCs alone (3×106 cells/mL) and NPCs+BMSCs (6×106 cells/mL; mixed 1:1) were cultured for 4 weeks in 1.2% alginate beads under base medium (BM), NCCM, or with addition of 10 ng/mL transforming growth factor-β1 (TGF-β1) as a positive control. Beads were assessed for glycosaminoglycan (GAG) and DNA contents by biochemical assays, GAG deposition by Alcian blue staining, and gene expression (aggrecan, versican, collagen 1 and 2, SOX9, A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and matrix metalloproteinase 13 [MMP13]) with real-time quantitative RT-PCR.>Results: NCCM increased NPC proliferation, proteoglycan production, and expression of genes associated with a healthy NP-like phenotype. BMSCs also showed increased proteoglycan production under NCCM, but these effects were not observed at the gene level. Combined stimulation of NPCs with NCCM and coculturing with BMSCs did not result in increased proteoglycan content compared to stimulation with NCCM alone.>Discussion: NCCM stimulates matrix production by both NPCs and BMSCs and directs NPCs toward a healthier phenotype. NCCM is therefore promising for IVD regeneration and identification of the bioactive components will be helpful to further develop this approach. In the current study, no synergistic effect of adding BMSCs was observed.
机译：>目标：先前已证明，来源于富含脊索细胞的髓核组织（NCCM）的条件培养基对混合的骨髓基质细胞（BMSC）和髓核细胞（NPC）具有刺激作用种体外细胞模型。当前研究的目的是评估NCCM对同源犬体外模型中NPC的刺激作用，并研究与NCCM联合刺激和添加BMSCs是否共同提供协同刺激作用。>方法： BMSCs和NPCs是从软骨营养不良的犬中收获的，证实了其早期椎间盘（IVD）变性。 NCCM由具有健康IVD的非软骨营养不良犬的NP组织产生。将单独的BMSC或NPC（3×10 6 细胞/ mL）和NPCs + BMSC（6×10 6 细胞/ mL; 1：1混合）培养4周在基础培养基（BM），NCCM或加入10μng/ mL转化生长因子-β1（TGF-β1）作为阳性对照的条件下，在1.2％海藻酸盐珠中加入。通过生化分析评估珠子的糖胺聚糖（GAG）和DNA含量，通过Alcian蓝染色评估GAG沉积，并评估基因表达（aggrecan，versican，胶原蛋白1和2，SOX9，A整合素和具有血小板反应蛋白基序5的金属蛋白酶（ADAMTS5），以及基质金属蛋白酶13 [MMP13]）。采用实时定量RT-PCR。>结果： NCCM可增加NPC增殖，蛋白聚糖生成以及与健康的NP样表型相关的基因的表达。 BMSCs还显示在NCCM下蛋白聚糖的产量增加，但在基因水平上未观察到这些作用。与单独使用NCCM刺激相比，结合使用NCCM刺激NPC和与BMSC共培养不会增加蛋白聚糖含量。>讨论： NCCM刺激NPC和BMSC共同产生基质，并指导NPC趋向更健康的表型。因此，NCCM有望用于IVD再生，生物活性成分的鉴定将有助于进一步开发这种方法。在当前的研究中，未观察到添加骨髓间充质干细胞的协同作用。

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