不同激活剂对人富血小板血浆形成凝胶及释放活性物质影响的实验研究  

Experimental research on the effects of different activators on the formation of platelet-rich gel and the release of bioactive substances in human platelet-rich plasma

作　　者：杨域 张卫[1] 程飚[1] 

机构地区：[1]南方医科大学附属广州军区广州总医院整形外科,510010

出　　处：《中华烧伤杂志》2017年第1期12-17,共6页Chinese Journal of Burns

基　　金：国家自然科学基金（81272105、81171812）;广东省科技计划项目（20148020212010）

摘　　要：目的探讨葡萄糖酸钙和凝血酶对人富血小板血浆（PRP）形成富血小板凝胶（PRG）与释放生物活性物质的影响以及临床意义。方法2016年5—8月，笔者单位招募符合入选标准的6名健康献血志愿者，采集每名志愿者血小板制备PRP。（1）每名志愿者取10 mL PRP，按照随机数字表法分为凝血酶激活组5 mL、葡萄糖酸钙激活组5 mL，凝血酶激活组加入100 U/mL凝血酶溶液0.5 mL、葡萄糖酸钙激活组加入100 g/L葡萄糖酸钙溶液0.5 mL，于37 ℃水浴中激活1 h。记录PRG形成时间，观察激活1 h内PRG的形成情况。激活1 h，收集PRG，一部分行HE染色观察纤维蛋白分布，一部分于透射电镜下观察血小板超微结构；收集上清液，采用ELISA法检测TGF-β1、血小板源性生长因子BB（PDGF-BB）、血管内皮生长因子、bFGF、EGF、胰岛素样生长因子Ⅰ的含量。（2）每名志愿者另取10 mL PRP同前分组，2次离心及2次PBS重新悬浮获得血小板悬液，同实验（1）加入相应激活剂处理1 h。采用纳米颗粒跟踪分析仪检测血小板来源不同直径微囊泡浓度及总微囊泡浓度。对数据行t检验。结果（1）凝血酶激活组PRG形成时间为（228±40）s，此时PRG体积最大；激活30 min PRG体积回缩至最小。葡萄糖酸钙激活组PRG形成时间为（690±71）s，此时PRG体积最大；激活55 min PRG体积回缩至最小。凝血酶激活组PRG形成时间明显短于葡萄糖酸钙激活组（t＝15.17，P〈0.01）。（2）HE染色显示，激活1 h，凝血酶激活组PRG中纤维蛋白红染面积大，密集分布；葡萄糖酸钙激活组PRG中纤维蛋白红染面积小，松散分布。透射电镜显示，激活1 h，凝血酶激活组PRG中血小板均呈碎片状；葡萄糖酸钙激活组PRG中可见正在裂解的血小板、α颗粒结构及未裂解的α颗粒、致密体结构。（3）凝血酶激活组PRP释放的PDGF-BB含量为（7.4±0.8）ng/mL，明显高于葡萄�ObjectiveTo explore the effects of calcium gluconate and thrombin on the formation of platelet-rich gel （PRG） and the release of bioactive substances in human platelet-rich plasma （PRP） and the clinical significance.MethodsSix healthy blood donors who met the inclusion criteria were recruited in our unit from May to August in 2016. Platelet samples of each donor were collected for preparation of PRP. （1） PRP in the volume of 10 mL was collected from each donor and divided into thrombin activation group （TA, added with 0.5 mL thrombin solution in dose of 100 U/mL） and calcium gluconate activation group （CGA, added with 0.5 mL calcium gluconate solution in dose of 100 g/L） according to the random number table, with 5 mL PRP in each group. Then the PRP of the two groups was activated in water bath at 37 ℃ for 1 h. The formation time of PRG was recorded, and the formation situation of PRG was observed within 1 hour of activation. After being activated for 1 h, one part of PRG was collected to observe the distribution of fibrous protein with HE staining, and another part of PRG was collected to observe platelet ultrastructure under transmission electron microscope （TEM）. After being activated for 1 h, the supernatant was collected to determine the content of transforming growth factor β1, platelet-derived growth factor BB （PDGF-BB）, vascular endothelial growth factor, basic fibroblast growth factor （bFGF）, epidermal growth factor, and insulin-like growth factorⅠby enzyme-linked immunosorbent assay. （2） Another 10 mL PRP from each donor was collected and grouped as above, and the platelet suspension was obtained after two times of centrifugation and resuspension with phosphate buffered saline, respectively. And then they were treated with corresponding activator for 1 h as that in experiment （1）. Nanoparticle tracking analyzer was used to detect the concentrations of microvesicles with different diameters and total microvesicles derived from platelet. Data were processed wit

关 键 词：富血小板血浆 生长因子 富血小板凝胶 微囊泡 

分 类 号：R969[医药卫生—药理学]