富血小板血浆对大鼠许旺细胞生物学行为的影响

Effect of platelet-rich plasma concentration on biological behavior of Schwann cells in vitro

目的观察不同浓度的富血小板血浆（PRP）对体外培养的许旺细胞（SCs）增殖、分泌功能及迁移的影响，探讨其促进周围神经再生的可能作用机制。方法从SD大鼠心脏穿刺取血，利用二次离心法制备PRP，对全血和PRP中血小板计数和血小板源性生长因子BB（PDGF—BB）和转化生长因子（TGF-B1）浓度测定；取3～5d龄的大鼠坐骨神经培养纯化SCs，将P1细胞分为实验组与对照组分别进行处理，实验组以含40．0％、20．0％、10．0％、5．0％和2．5％PRP的条件培养液干预，并设立空白对照组。于干预不同时间点采用CCK-8法测定SCs增殖活性情况，用实时荧光定量PCR方法检测细胞神经生长因子（NGF）和胶质细胞源神经营养因子（GDNF）mRNA表达的变化，ELISA检测SCs分泌NGF和GDNF的水平，Transwell小室检测各组SCs的迁移能力。结果PRP血小板回收率达65％，PDGF．BB和TGF-β1浓度明显高于血清（P〈0．01）；与空白对照组相比，低于20．0％浓度的PRP呈浓度依赖性促进SCs增殖和迁移，而40．0％浓度组细胞增殖和迁移受到抑制；SCs分泌的NGF和GDNF和其mRNA表达均较对照组明显增加，同样在低于20．0％浓度的范围内呈现量效关系，40．0％浓度组则显示抑制作用。结论PRP在适当浓度范围可以促进SCs的分裂增殖，合成分泌NGF和GDNF以及迁移的能力，具有潜在的促周围神经再生的作用。

Objective To investigate the effect of platelet-rich plasma（PRP） concentration on SCs in order to determine the plausibility of using this plasma-derived therapy for peripheral nerve injury. Methods PRP was obtained from rats by double-step centrifugation and was characterized by determining platelet num-bers, platelet-derived growth factor-BB （PDGF-BB） and transforming growth factor-β1 （TGF-β1） concentra-tions. Primary cultures of rat SCs obtained from sciatic nerves of neonatal rats were exposed to various concentra-tions of PRP （40.0%, 20. 0%, 10. 0%, 5.0% and 2. 5% ）. Cell proliferation assays were performed to study to assess SCs proliferation. Quantitative real-time PCR and ELISA analysis were performed to determine the abil-ity of PRP to induce SCs to secreteuct nerve growth factor （NGF） and glial cell line-derived neurotrophic factor （GDNF）. Microchemotaxis assay was used to analyze the cell migration capacity. Results The results ob- tained indicated that the platelet concentration, PDGF-BB and TGF-β1 in our PRP preparations were significantly higher than in whole blood. Cell culture experiments showed that 20. 0%-2. 5% PRP significantly stimulated SCs proliferation and migration compared to untreated controls in a dose-dependent manner. In addition, the ex- pression and secretion of NGF and GDNF were significantly increased. However, the above effects of SCs were suppressed by at high PRP concentrations （40. 0% ）. Conclusion The appropriate concentration of the PRP has the potency to stimulate cell proliferation, induced the synthesis of neurotrophic factors, and significantly in- creased migration of SCs dose dependently.