[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].

Objective: To 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. Methods: Six 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 β(1, )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 with t test. Results: (1) The formation time of PRG in group TA was (228±40) s, and the PRG volume reached the maximum at this moment. The PRG volume shrunk to the minimum after 30 minutes of activation. The formation time of PRG in group CGA was (690±71) s, and the PRG volume reached the maximum at this moment. After 55 minutes of activation, the PRG volume shrunk to the minimum. The formation time of PRG in group TA was obviously shorter than that in group CGA (t=15.17, P<0.01). (2) HE staining showed that after 1 hour of activation, the red-stained area of fibrous protein in PRG of group TA was large and densely distributed, while that of group CGA was small and loosely distributed. TEM revealed that after 1 hour of activation, the platelets in PRG of group TA were fragmented, while lysing platelet structure, lysing α granule structure, intact α granule structure, and intact dense body structure were observed in PRG of group CGA. (3) The content of PDGF-BB released by PRP in group TA was (7.4±0.8) ng/mL, which was obviously higher than that in group CGA [(4.9±0.5) ng/mL, t=5.41, P<0.01]. The content of bFGF released by PRP in group CGA was (960±151) pg/mL, which was significantly higher than that in group TA [(384±56) pg/mL, t=8.75, P<0.01]. The content of the other 4 growth factors released by PRP in the two groups was close (with t values from 0.11 to 1.97, P values above 0.05). (4) The concentrations of total microvesicles, microvesicles with diameter more than 100 nm, and exosomes with diameter less than or equal to 100 nm derived from platelet in group CGA were (165.8±15.1)×10(8)/mL, (142.4±12.3)×10(8)/mL, and (23.4±2.9)×10(8)/mL respectively, which were significantly higher than those in group TA [(24.7±4.6)×10(8)/mL, (22.6±4.0)×10(8)/mL, and (2.1±0.7)×10(8)/mL, with t values from 17.36 to 22.66, P values below 0.01]. Conclusions: Calcium gluconate can slowly activate PRP, resulting in slowly shrunk PRG with high content of bFGF and high concentration of microvesicles, which is suitable for repairing articular cavity and sinus tract wound. Thrombin can rapidly activate PRP, resulting in quickly shrunk PRG with high content of PDGF-BB and a certain concentration of microvesicles, which is suitable for repairing acute trauma.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-β(1)、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，明显高于葡萄糖酸钙激活组[(4.9±0.5)ng/mL，t＝5.41，P<0.01]。葡萄糖酸钙激活组PRP释放的bFGF含量为(960±151)pg/mL，明显高于凝血酶激活组[(384±56)pg/mL，t＝8.75，P<0.01]。2组PRP释放的其余4种生长因子含量相近(t值为0.11～1.97，P值均大于0.05)。(4)葡萄糖酸钙激活组血小板来源的总微囊泡、直径大于100 nm微囊泡、直径小于或等于100 nm外泌体的浓度分别为(165.8±15.1)×10(8)/mL、(142.4±12.3)×10(8)/mL、(23.4±2.9)×10(8)/mL，均明显高于凝血酶激活组[分别为(24.7±4.6)×10(8)/mL、(22.6±4.0)×10(8)/mL、(2.1±0.7)×10(8)/mL，t值为17.36～22.66，P值均小于0.01]。 结论： 葡萄糖酸钙缓慢激活PRP，形成的PRG回缩缓慢、释放高含量的bFGF和高浓度的微囊泡，宜于修复关节腔及窦道性创面；凝血酶快速激活PRP，形成的PRG回缩较快，释放高含量的PDGF-BB和一定浓度的微囊泡，宜于修复急性创伤。.