去白细胞和富白细胞富血小板血浆对软骨细胞增殖迁移的作用

Effects comparison of pure platelet-rich plasma and leukocyte- platelet-rich plasma on chondrocyte proliferation and migration

目的探讨去白细胞富血小板血浆(P-PRP)和富白细胞富血小板血浆(L-PRP)对软骨细胞增殖和迁移的作用,为临床合理使用富血小板血浆治疗骨关节炎提供依据。方法 2015年6月至8月,上海交通大学附属第六人民医院骨科共招募20名健康成年献血志愿者,随机数法将其分为两组后分别采集全血制备P-PRP和L-PRP,分别为P-PRP组和L-PRP组。在测定P-PRP和L-PRP中的白细胞、血小板、血小板源性生长因子AB(PDGF-AB)和转化生长因子β1(TGF-β1)、白介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)浓度后,使用其处理人软骨细胞。通过CCK-8实验和Transwell实验分析两者对软骨细胞增殖和迁移的作用。P-PRP和L-PRP之间细胞和细胞因子浓度的差异使用分独立样本t检验分析;胎牛血清(FBS)、P-PRP和L-PRP之间对软骨细胞增殖和迁移作用的差异使用单因素方差分析及Bonferroni事后检验分析。结果 P-PRP组和L-PRP组中的血小板、PDGF-AB和TGF-β1浓度差异无统计学意义,但是两组之间白细胞、IL-1β和TNF-α浓度的差异则有统计学意义(白细胞浓度:t=8.095,P<0.01;IL-1β浓度:t=5.342,P<0.01;TNF-α浓度:t=5.297,P<0.01),均以L-PRP组为高。单因素方差分析提示,FBS组、P-PRP组和L-PRP组之间对软骨细胞增殖和迁移的影响有统计学意义(增殖:F=29.37,P<0.01;迁移:F=87.54,P<0.01);而Bonferroni事后检验则进一步提示,虽然与FBS相比,P-PRP和L-PRP对软骨细胞增殖和迁移均有促进作用(P-PRP组与FBS相比:增殖P<0.001,迁移P<0.01;L-PRP组与FBS相比:增殖P=0.010,迁移P<0.01),但是两组之间对软骨细胞增殖和迁移作用的差异有统计学意义(增殖P=0.005,迁移P=0.011),P-PRP的效果优于L-PRP。结论 L-PRP中的白细胞可以通过分泌炎症因子对其治疗骨关节炎的疗效产生不利影响。白细胞和炎症因子浓度较低的P-PRP可能更适用于骨关节炎的临床治疗。

Objective To evaluate the effects of leukocytes in platelet-rich plasma （PRP） on cartilage regeneration by comparing the effects of pure platelet-rich plasma （P-PRP） and leukocyte- platelet-rich plasma （L-PRP） on chondrocyte proliferation and migration. Methods Between June and August 2015, 20 healthy adult volunteers were recruited to donate whole blood for P-PRP and L-PRP preparation. P-PRP and L-PRP were prepared and measured for concentrations of leukocytes, platelets, platelet-derived growth factor AB （PDGF-AB） , transforming growth factor β1 （TGF-β1）, interleukin 1 β（IL-1β）, and tumor necrosis factor a （TNF-α）. The human articular chondrocytes were treated with 10% fetal bovine serum （FBS） , P-PRP, or L-PRP, and the effects of P-PRP and L-PRP on human articularchondrocytes were evaluated by CCK-8 assay and Transwell assay, respectively. The differences between L- PRP and P-PRP in cellular and cytokine components were analyzed by Students＇ t test, and the differences among FBS, L-PRP and P-PRP with regards to the effects of them on cell proliferation and migation were analyzed by one-way analysis of variance and Bonferroni post-hoc test. Results No significant difference was found between P-PRP and L-PRP in concentrations of platelets and growth factors. However, significant differences were found between P-PRP and L-PRP with regards to concentrations of leukocytes （t= 8.095, P〈0.01）, IL-1β （t = 5.342, P 〈0.01）; TNF-α （t = 5.297, P〈0.01）, and L-PRP showed higher concentrations of these components. Results of CCK-8 and Transwell assays demonstrated that both P-PRP and L-PRP promoted chondrocyte proliferation and migration compared with FBS （ chondrocyte proliferation： F = 29.37, P 〈 0. 01 ; ehondroeyte migration ： F = 87. 54, P 〈 0. 01 ） , but P- PRP showed greater effects than L-PRP （P = 0.005 for chondrocyte proliferation, and P = 0.011 for chondroeyte migration）. Conclusion Concentrated leukoeytes in L-PRP may release high levels of pro- inflammatory cytokines to decrease the beneficial effects of growth factors on osteoarthritic cartilage, indieating that P-PRP may be more suitable for the treatment of osteoarthritis.