冷冻保存富血小板血浆对创伤修复的影响及体外机制研究

The impact and in vitro mechanisms of frozen stored platelet-rich plasma on wound repair

目的评估-80℃超低温冷冻保存后冻融的富血小板血浆(PRP)对伤口愈合相关细胞的影响,揭示超低温冻融的PRP在创伤修复中的机制,明确低温冷冻保存PRP对促进伤口愈合应用的可行性和有效性。方法使用未激活的新鲜PRP、钙离子激活的新鲜PRP和冻融后PRP分别与巨噬细胞、成纤维细胞和血管形成细胞共培养,测定比较巨噬细胞极化与炎症反应相关因子的表达及细胞迁移率和增殖率等多项指标,分析PRP对巨噬细胞极化、炎症及细胞增殖的影响。结果与对照组比较,超低温冻融后的PRP使巨噬细胞M1样极化基因iNOS表达降低(P<0.0001),NO分泌减少(P<0.001),尿素含量增加(P<0.0001),M1相关炎性因子肿瘤坏死因子-α(TNF-α)降低(P<0.001),白细胞介素(IL)-1分泌降低(P<0.001),M2相关抗炎因子IL-10、IL-12分泌水平增加(P<0.01,P<0.05)。此外,冻融PRP共培养明显促进细胞迁移,提高了血管形成效率,高于新鲜PRP组(P<0.01),与激活PRP组效果相当。细胞活死染色和CCK-8增殖实验也显示与冻融PRP共培养的L929和HUVEC细胞增殖率均显著增加(P<0.01)。结论-80℃超低温冷冻保存后的PRP能够显著增强细胞的迁移、分化和增殖能力,同时抑制炎症因子产生,促进巨噬细胞的M2样极化。低温冷冻保存可被视为一种有效的PRP保存方法。

Objective To evaluate the effects and mechanisms of frozen-thawed platelet-rich plasma(PRP)stored at-80℃on wound healing-related cells.Furthermore,to explore the feasibility and effectiveness of using cryopreserved PRP at low temperatures for promoting wound healing.Methods Using non-activated fresh PRP,calcium-activated fresh PRP,and post-thawed PRP,co-cultured with macrophages,fibroblasts,and vascular endothelial cells,the study measured and compared the expression of polarization and inflammatory factors associated with macrophages,as well as cell migration and proliferation rates,among other indicators,to analyze the effects of PRP on macrophage polarization,inflammation,and cell proliferation.Results Compared with the control group,post-cryopreserved PRP at ultra-low temperatures resulted in decreased expression of M1-like polarized macrophage gene iNOS(P<0.0001),reduced NO secretion(P<0.001),increased urea content(P<0.0001),decreased M1-related inflammatory factor tumor necrosis factor-alpha(TNF-α)(P<0.001),and decreased secretion of white blood cell interleukin(IL)-1(P<0.001);M2-related anti-inflammatory factor IL-10 secretion levels increased(P<0.01),and IL-12 secretion increased(P<0.05)Furthermore,co-culturing with frozen-thawed PRP significantly promoted cell migration and enhanced vascular formation efficiency,surpassing the effects of fresh PRP and being comparable to activated PRP(P<0.01).Cell viability assays and CCK-8 proliferation experiments also showed a significant increase in the proliferation rates of L929 and HUVEC co-cultured with frozen-thawed PRP(P<0.01).Conclusion After being cryopreserved at-80℃,PRP has been proven to significantly enhance cell migration,differentiation,and proliferation capabilities,while inhibiting the production of inflammatory factors and promoting M2 polarization of macrophages.Therefore,low-temperature cryopreservation can be considered as an effective method for PRP preservation.