浓缩血小板的研究进展

1、解 放 军 总 医 院 第 一 附 属 医 院解 放 军 总 医 院 第 一 附 属 医 院烧 伤 整 形 三 科 暨 创 面 修 复 中 心烧 伤 整 形 三 科 暨 创 面 修 复 中 心 赵帆赵帆 郝岱峰郝岱峰内容l血小板简介及其功能l浓缩血小板及其应用人群的选择l浓缩血小板的制备方法及分类l浓缩血小板的激活及使用方法l浓缩血小板在创面修复中的作用l目前研究中存在的问题血小板简介及功能血小板血小板是血细胞中的一种，它是从骨髓巨核细胞的细胞质脱落的的具有生物活性的小块胞质，体积小，无细胞核，呈双面微凸的圆盘状。生理特性：粘附、聚集、释放、收缩粘附、聚集、释放、收缩基本功能：止血凝血、维持血

2、管内皮的完整性止血凝血、维持血管内皮的完整性 Platelet ultrastructure. 14浓缩血小板浓缩血小板（浓缩血小板（Platelets ConcentratePlatelets Concentrate）根据其临床用途可分为两类：两类：第一类第一类：体内，是外科常用的成分输血制品，浓缩血小板一般用于血小板减少，失血症状明显而又需手术者。第二类第二类：体外，常用于局部软组织和骨组织的修复，主要是应用血小板内还有丰富的细胞因子，它们在参与创面的修复过程中发挥重要的作用。目前文献中常见的有四种浓缩血小板类型，也可称为Platelet-Rich Preparation应用人群的选择 明

3、确有传染病的患者，如乙肝、梅毒、艾滋等； 肿瘤患者； 服用血小板抑制剂的或血小板功能存在障碍的患者；如口服阿司匹 林、氯吡格雷等； 血小板减少的患者；制备临床上常用的浓缩血小板主要通过两种两种方法制备商业的血小板分离系统商业的血小板分离系统手工分离手工分离制备过程中的影响因素众多，主要包括以下几方面：患者之间的个体差异患者之间的个体差异血液的采集血液的采集：无菌原则、抗凝剂的选择（ACD-A/CPD）、抗凝剂和血液的比例（1:10）离心次数离心次数： 单次离心和二次离心转速转速/ /离心力离心力： Hard and Soft centrifugation 离心时间离心时间离心温度离心温度 商业

4、分离系统Commercial SystemBlood Vol(ml)Centrifugation(No.of Spins)PRP Vol (ml)Platelet ConcentrationActivator(+/-)Activator:PRPLeukocytes(+/-)Cascade 9-1814-91-1.5CaCl21:10-GPS 601109.3Trombin1:10+ACP9132-3None1:10-Smart PRP220-12023-204-6Trombin1:10+PRGF9-7214-322-3CaCl21:10-Magellan30-60263-9CaCl21:10+

5、Angel40241-18None1:10+/-Genesis CS30-6014-109CaCl21:10NSSequire50251.6Trombin Bovine1:10NSPlatelex5024-6NSBatroxobin1:10+Symphony PCS55-1101NS3-6Thrombin1:10NSJP200020215-9CaCl2 and Thrombin1:10+GLO PRP8.520.65-6CaCl2 and Thrombin1:10+KYOCERA20227-8CaCl21:10+Selphyl8120.5CaCl21:10+MyCells10114-5CaCl

6、2 and Thrombin1:10+Dr.Shins System8.5113-4CaCl2 and Thrombin1:10+PCCS手工分离 Flow chart describing preparation of PRP. 2浓缩血小板分类 L-PRP PRP P-PRP 浓缩血小板类型 PRGF L-PRF PRF R-PRF10PRPPRP. Classical manual platelet-rich plasma (PRP) protocol using a two-step centrifugation procedure. 311PRGFPRGF Commonly desc

7、ribed protocol for Anituas PRGF. 3PPGF: plasma poor in growth factorsPRGF: plasma rich in growth factors12PRFPRF Choukrouns platelet-rich fibrin (PRF) method . 313Structural DifferencesSchematic illustration of the matrix and cell architecture of the four categories of platelet concentrates. 3Activa

8、ted versus Nonactivated浓缩血小板的激活15 Activated浓缩血小板的激活方式应该考虑以下三个三个方面：l 血小板激活剂血小板激活剂的种类 牛凝血酶、人凝血酶、钙剂、凝血酶受体激活肽（TRAP）、胶原、类凝血酶-巴曲酶、反复冻融、壳聚糖、纳秒脉冲电场（nsPEF） ；l 激活剂的浓度的选择l 激活剂和浓缩血小板的比例 影响细胞因子的释放时间影响细胞因子的释放时间 影响细胞因子的释放量影响细胞因子的释放量16纳秒脉冲电场（纳秒脉冲电场（Nanosecond Pulse Electric Field， nsPEF）ActivatorRepresentative volt

9、age and current tracing for pulse electric field condition used to activate PRP. Electrical signals were measured on a 2mm cuvette containing PRP and stimulated with pulse electric field for PRP activation. Each sample was e x p o s e d t o f i v e p u l s e s , o f approximately 500 nanoseconds pul

10、se width, 4 kV peak voltage and more than 300 A peak current per pulse .417Non-activatedDiabetic mouse full-thickness wound healing .518Non-activatedEffects of platelet treatment on fibroblast cultures .5Non-activatedIn vitro three-dimensional tissue defect assay .520Activated versus NonactivatedSch

11、ematic depiction of the effect of physiologic and unspecific activation of platelets on wound healing .421在创面修复中的作用Cytokines/ChemokineIL-6IL-8PF-4MIP-1RANTESFractalkineNAP2GRO-ENA-78sCD40LSDF-1Growth FactorPlatelet Derived Growth Factor, PDGF-AB/BBTransforming Growth Factor-beta, TGF-1/2Vascular End

12、othelial Growth Factor, VEGFEpidermal Growth Factor, EGFBasic Fibroblast Growth Factor, bFGFInsulin like Growth Factor (IGF 1 and 2)Connective Tissue Growth Factor (CTGF)Keratinocyte Growth Factor (KGF)Hepatocyte Growth Factor(HGF)Platelet Derived Endothelial Growth Factor (PDEGF)Platelet Derived An

13、giogenic Factor(PDAF)生长因子在创面修复中的作用Schematic illustration of the role of GFs during the different stages of the wound healing process .623细胞因子在创面修复中的作用This is true for bacteria like MSSA, Group A Streptococcus, and Neisseria gonorrhoeae as well.7. 目前存在的问题缺乏统一的浓缩血小板的制备标准临床应用时能否激活仍存在争议，若激活，激活剂种类、浓度及其与浓

14、缩血小板的比例如何确定无法确定有临床疗效的浓缩血小板中最适的血小板的浓度参考文献1. Yeaman MR. The role of platelets in antimicrobial host defense. Clin Infect Dis 1997 25(5): 951-968; quiz 969-9702. Dhurat R Sukesh M. Principles and Methods of Preparation of Platelet-Rich Plasma: A Review and Authors Perspective. J Cutan Aesthet Surg 2014

15、 7(4): 189-1973. Dohan Ehrenfest DM, Rasmusson L Albrektsson T. Classification of platelet concentrates: from pure platelet-rich plasma (P-PRP) to leucocyte- and platelet-rich fibrin (L-PRF). Trends Biotechnol 2009 27(3): 158-1674. Scherer SS, Tobalem M, Vigato E et al. Nonactivated versus thrombin-

16、activated platelets on wound healing and fibroblast-to-myofibroblast differentiation in vivo and in vitro. Plast Reconstr Surg 2012 129(1): 46e-54e5. Frelinger Iii AL, Torres AS, Caiafa A et al. Platelet-rich plasma stimulated by pulse electric fields: Platelet activation, procoagulant markers, growth factor release and cell proliferation. Platelets 20151-86. Everts PA, Knape JT, Weibrich G et al. Platelet-rich plasma