冰冻保存PRP对IL-1β诱导的软骨细胞炎症反应的影响

Effect of Frozen Storage Platelet-rich Plasma on Inflammatory Response of IL-1β-I Induced Chondrocyte

目的探讨冰冻保存对富血小板血浆(platelet-rich plasma,PRP)释放生长因子及体外抗软骨细胞炎症作用的影响,为合理利用自体PRP技术,保证临床治疗效果提供实验依据。方法以机采血小板为原料,用4种不同方式冰冻保存,分别为–20℃、–80℃直接冻存及加5%DMSO–20℃、–80℃冻存,3个月后解冻制备PRP凝胶。ELISA法检测凝胶上清液中PDGF-AB、VEGF和IGF-1含量。制备体外软骨炎症细胞模型,CCK-8法检测冻存PRP对其增殖的影响,ELISA检测PRP对软骨炎症细胞分泌炎症因子IL-6的影响。结果与新鲜PRP组相比,冷冻保存的PRP制备的凝胶上清液中PDGF-AB含量无显著变化,而VEGF、IGF-1含量明显下降。CCK-8结果表明,加5%DMSO–80℃冻存的PRP,对软骨炎症模型细胞增殖有显著促进作用,效果与新鲜制备的PRP相当,而–20℃、–80℃直接冻存以及加DMSO保护剂–20℃冻存的PRP对软骨炎症模型细胞增殖没有促进作用。检测软骨炎症模型细胞培养上清液中细胞因子IL-6的含量发现,PRP以四种冻存方式冻存3个月后,和新鲜PRP相比,在抑制软骨炎症细胞分泌炎症细胞因子IL-6方面无显著性差异。结论冻存之后PRP凝胶上清液中部分细胞因子含量减少,但在体外针对IL-1β诱导的软骨细胞损伤,冻存PRP和新鲜PRP有相同的保护作用。

Objective The aim of the present study is to investigate whether frozen could influence platelet-rich plasma(PRP)releases growth factors,and whether frozen PRP could protect chondrocyte from IL-1β-induced injury.Methods PRP concentrates were obtained from healthy subjects by apheresis and were divided into 4 groups,then were frozen for as long as 3 months.The storage condition was–20℃without preservation agent,–80℃without preservation agent,–20℃in 5%DMSO and–80℃in 5%DMSO.The growth factors(PDGF-AB,VEGF and IGF-1)in PRP releasates were quantified by ELISA.The effect of PRP on the proliferation of IL-1βinduced chondrocytes’inflammation was detected by CCK-8.The levels of IL-6 in cells were detected by ELISA.Results No significant difference was observed in PDGF-AB levels among fresh PRP group and frozen PRP groups.VEGF and IGF-1 levels were significantly decreased in the PRP samples stored at frozen conditions.No obvious difference with CCK-8 assay was observed between the PRP stored in–80℃in 5%DMSO group and fresh PRP group.They both can promote IL-1β-induced inflammatory chondrocyte's proliferation.The other three experimental groups can’t promote IL-1β-induced chondrocyte proliferation.After PRP treatment,IL-6 production of inflammatory chondrocyte was decreased.No statistically significant differences were found in IL-6 production between fresh PRP and frozen PRPs.Conclusion Some growth factor levels of PRP were reduced after frozen storage.But the frozen PRP can protect IL-1β-induced chondrocyte injury by inhibiting inflammatory response.