Bousnaki M, Bakopoulou A, Pich A, Papachristou E, Kritis A, Koidis P
Department of Prosthodontics, School of Dentistry, Faculty of Health Sciences (FHS), Aristotle University of Thessaloniki (AUTh), GR-54124, Thessaloniki, Greece.
Research Core Unit Proteomics & Institute of Toxicology, Hannover Medical School, 30625, Hannover, Germany.
Stem Cell Rev Rep. 2022 Apr;18(4):1372-1407. doi: 10.1007/s12015-021-10255-2. Epub 2021 Sep 22.
There is substantial evidence supporting the anti-inflammatory and regenerative potential of dental pulp stem cells (DPSCs) through direct cell transplantation or paracrine action. However, DPSC secretome profile remains inadequately studied. This study provides proteomic profiling of the human DPSC secretome by comparatively analysising cell lysates and respective culture supernatants (i.e. conditioned media-CM) under variable oxygen tension conditions (normoxia-20% O/CM_Norm vs. hypoxia 2% O/CM_Hyp) and/or stimulation with Tumor Necrosis Factor alpha (TNF-α). DPSC-CM samples and respective crude lysates (DPSC-CL) were collected and subjected to SDS-PAGE, followed by LC-MS/MS analysis. The identified proteins were analyzed by Gene Ontology, Reactome, and String databases. The anti-inflammatory properties of DPSC-CMs were validated via an in vitro RAW_246.7 murine macrophages model through evaluation of the expression of pro-and anti-inflammatory markers by real-time PCR. Results showed a total of 2413 proteins identified in CM_Norm, 2479 in CM_Norm+TNF-α, 1642 in CM_Hyp, and 2002 in CM_Hyp + TNF-α samples. CM_Norm contained 122 proteins statistically significantly upregulated compared to the CM_Hyp and involved in pathways related to "ECM organization", "cellular response to hypoxia", and "IL signaling". Functional network analysis showed that TGFβ1, TIMP1 and TIMP2 were key nodes among proteins significantly upregulated in the CM_Norm compared to the CM_Hyp, interacting with more than 10 proteins, each. DPSC-CM application in the in vitro RAW_246.7 model decreased the expression of pro-inflammatory markers (MMP-3, MMP-9, MMP-13, MCP-1), while increasing anti-inflammatory markers (IL-10). Overall, DPSC-CM collected under normoxic conditions is enriched with anti-inflammatory, tissue repair and regenerative factors, which prompts further investigation on its therapeutic applications.
有大量证据支持牙髓干细胞（DPSCs）通过直接细胞移植或旁分泌作用发挥的抗炎和再生潜力。然而，DPSC分泌组概况仍未得到充分研究。本研究通过比较分析在可变氧张力条件下（常氧-20% O₂/CM_Norm与低氧2% O₂/CM_Hyp）和/或用肿瘤坏死因子α（TNF-α）刺激后的细胞裂解物和相应的培养上清液（即条件培养基-CM），对人DPSC分泌组进行蛋白质组分析。收集DPSC-CM样本和相应的粗裂解物（DPSC-CL），进行SDS-PAGE，随后进行LC-MS/MS分析。通过基因本体论、反应体和字符串数据库对鉴定出的蛋白质进行分析。通过实时PCR评估促炎和抗炎标志物的表达，在体外RAW_246.7小鼠巨噬细胞模型中验证了DPSC-CM的抗炎特性。结果显示，在CM_Norm样本中鉴定出2413种蛋白质，在CM_Norm+TNF-α样本中鉴定出2479种，在CM_Hyp样本中鉴定出1642种，在CM_Hyp+TNF-α样本中鉴定出2002种。与CM_Hyp相比，CM_Norm含有122种在统计学上显著上调的蛋白质，这些蛋白质参与了与“细胞外基质组织”、“细胞对低氧的反应”和“白细胞介素信号传导”相关的途径。功能网络分析表明，与CM_Hyp相比，TGFβ1、TIMP1和TIMP2是CM_Norm中显著上调的蛋白质中的关键节点，各自与10多种蛋白质相互作用。在体外RAW_246.7模型中应用DPSC-CM可降低促炎标志物（MMP-3、MMP-9、MMP-13、MCP-1）的表达，同时增加抗炎标志物（IL-10）。总体而言，在常氧条件下收集的DPSC-CM富含抗炎、组织修复和再生因子，这促使对其治疗应用进行进一步研究。
