The Key Laboratory of Laboratory Medical Diagnostics in the Ministry of Education and Department of Clinical Biochemistry, College of Laboratory Medicine, 12550Chongqing Medical University, Chongqing, China.
Department of Endocrinology, Multidisciplinary Diabetic Foot Medical Center, 159412Chongqing Emergency Medical Center, Chongqing University Central Hospital, Chongqing University, Chongqing, China.
Cell Transplant. 2021 Jan-Dec;30:9636897211017833. doi: 10.1177/09636897211017833.
PRP-Exos are nanoscale cup-shaped vesicles that carry a variety of proteins, mRNAs, microRNAs, and other bioactive substances. PRP-Exos can be formed through several induction pathways, which determine their molecular profiles and facilitate their tailormade participation in intercellular communication. Currently, little is known on how the PRP-Exos activation method influences the quality and quantity of PRP-Exos. The present study aims to observe and analyze the number, profile, and growth factors of PRP-Exos through TEM, Nanoflow, and WB after PRP activation and compare the difference in function of PRP-Exos on HUVECs, with different stimuli (calcium gluconate, thrombin, or both). We found that PRP activated with both thrombin and calcium gluconate harvested the highest concentration of exosomes [(7.16 ± 0.46) × 10 particles/ml], compared to thrombin group [(4.87 ± 0.15) × 10 particles/ml], calcium gluconate group [(5.85 ± 0.43) × 10 particles/ml], or saline group [(7.52 ± 0.19) × 10 particles/ml], respectively ( < 0.05) via Nanoflow analysis. The WB analysis showed that cytokines (VEGF, PDGFBB, bFGF, TGF-β) are differentially encapsulated in PRP-Exos, depending on the PRP stimulus, in which the mixture-PRP-Exos yielded the highest concentration of cytokines. In the function assay of PRP-Exos on HUVECs, the mixture-PRP-Exos promoted HUVECs proliferation, increased HUVECs migration, promoted the formation of vessel-like by HUVECs via the AKT ERK signal pathway more dramatically, compared with other groups. In summary, our studies showed that PRP activated by the mixture of calcium gluconate and thrombin harvested the best quality of exosomes which had the top biological functions. This study provides a protocol for selecting appropriate PRP activators to obtain high-quality exosomes for future applications.
PRP-Exos 是纳米级杯状囊泡，携带多种蛋白质、mRNA、microRNA 和其他生物活性物质。PRP-Exos 可以通过几种诱导途径形成，这些途径决定了它们的分子谱，并促进它们有针对性地参与细胞间通讯。目前，对于 PRP-Exos 的激活方法如何影响 PRP-Exos 的质量和数量知之甚少。本研究旨在通过 TEM、Nanoflow 和 WB 观察和分析 PRP 激活后 PRP-Exos 的数量、谱图和生长因子，并比较不同刺激物（葡萄糖酸钙、凝血酶或两者）对 HUVECs 上 PRP-Exos 功能的差异。我们发现，用凝血酶和葡萄糖酸钙激活的 PRP 收获的外泌体浓度最高[(7.16±0.46)×10 个颗粒/ml]，与凝血酶组[(4.87±0.15)×10 个颗粒/ml]、葡萄糖酸钙组[(5.85±0.43)×10 个颗粒/ml]或生理盐水组[(7.52±0.19)×10 个颗粒/ml]相比，差异有统计学意义(<0.05)。WB 分析显示，细胞因子(VEGF、PDGFBB、bFGF、TGF-β)根据 PRP 刺激物的不同，被差异包裹在 PRP-Exos 中，其中混合物-PRP-Exos 产生的细胞因子浓度最高。在 PRP-Exos 对 HUVECs 的功能检测中，与其他组相比，混合物-PRP-Exos 更显著地促进了 HUVECs 的增殖，增加了 HUVECs 的迁移，通过 AKT-ERK 信号通路促进了 HUVECs 血管样形成。综上所述，我们的研究表明，用葡萄糖酸钙和凝血酶混合物激活的 PRP 收获了最好质量的 exosomes，具有最佳的生物学功能。本研究为选择合适的 PRP 激活剂提供了方案，以获得高质量的 exosomes 用于未来的应用。
