CAJ | 학술논문

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-β1)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.
목적 관찰불동농도적부혈소판혈장(PRP)대체외배양적허왕세포(SCs)증식、분비공능급천이적영향,탐토기촉진주위신경재생적가능작용궤제. 방법 종SD대서심장천자취혈,이용이차리심법제비PRP,대전혈화PRP중혈소판계수화혈소판원성생장인자BB (PDGF-BB)화전화생장인자(TGF-β1)농도측정；취3～5d령적대서좌골신경배양순화SCs,장P1세포분위실험조여대조조분별진행처리,실험조이함40.0％、20.0％、10.0％、5.0％화2.5％ PRP적조건배양액간예,병설립공백대조조.우간예불동시간점채용CCK-8법측정SCs증식활성정황,용실시형광정량PCR방법검측세포신경생장인자(NGF)화효질세포원신경영양인자(GDNF) mRNA표체적변화,ELISA검측SCs분비NGF화GDNF적수평,Transwell소실검측각조SCs적천이능력. 결과 PRP혈소판회수솔체65％,PDGF-BB화TGF-β1농도명현고우혈청(P＜0.01)；여공백대조조상비,저우20 0％농도적PRP정농도의뢰성촉진SCs증식화천이,이40.0％농도조세포증식화천이수도억제；SCs분비적NGF화GDNF화기mRNA표체균교대조조명현증가,동양재저우20.0％농도적범위내정현량효관계,40.0％농도조칙현시억제작용.결론 PRP재괄당농도범위가이촉진SCs적분렬증식,합성분비NGF화GDNF이급천이적능력,구유잠재적촉주위신경재생적작용.
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 numbers,platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β1 (TGF-β1) concentrations.Primary cultures of rat SCs obtained from sciatic nerves of neonatal rats were exposed to various concentrations 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 ability of PRP to induce SCs to secretcuct ncrve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF).Microchemotaxis assay was used to analyze the cell migration capacity.Results The results obtained 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 expression 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 increased migration of SCs dose dependently.