摘要目的 对比分析不同激活剂对富血小板血浆(PRP)中转化生长因子-β1(TGF-β1)及血小板源性生长因子(PDGF-AB)释放曲线的影响.方法 抽取10名健康成年志愿者外周静脉全血36 ml,采用二次离心法自制PRP;按1000 U凝血酶溶于1 ml 10％氯化钙溶液制备血小板激活剂.按10:1的容量比将PRP与激活剂混合(凝血酶-PRP组);按10:1的容量比将PRP与10％氯化钙混合(氯化钙-PRP组);分别以新鲜全血(全血组)以及未加血小板激活剂的PRP(PRP组)为对照组,将上述4组在37℃温水中孵育0、1、8、24、72和168 h,采用ELISA法测定各组不同时间点的TGF-β1和PDGF-AB浓度,绘制TGF-β1和PDGF-AB的释放曲线,并采用重复测量方差分析比较不同组别中TGF-β1和PDGF-AB的释放曲线.结果 (1)全血组与PRP组中TGF-β1和PDGF-AB在168 h内随着时间的推移而持续增高.PRP在与凝血酶混合后,立即形成凝胶状,TGF-β1和PDGF-AB均立即明显升高,在激活后1 h达到高峰,TGF-β1和PDGF-AB分别由(42±21)和(77±18)μg/L增至高峰(84±21)和(124±35)μg/L,随后逐渐下降,释放曲线直接而快速;而PRP与氯化钙混合后,约1 h才形成凝胶状,TGF-β1和PDGF-AB均缓慢持续增高,可在168 h内一直保持较高水平.(2)PRP组中TGF-β1和PDGF-AB的AUC0-168h均高于全血组(均P<0.05),氯化钙-PRP组中TGF-β1的AUC0-168h高于凝血酶-PRP组(Z=-2.26,P<0.05),但氯化钙-PRP组与凝血酶-PRP组中PDGF-AB的AUC0-168h比较差异无统计学意义(Z=-1.512,P=0.131).结论 使用氯化钙作为激活剂时富血小板血浆中TGF-β1和PDGF-AB释放浓度较高,释放时间较长,曲线下面积最大.

abstractsObjective To compare and analyze the effects of different activators on the release curve of TGF-β1 and PDGF-AB in platelet rich plasma(PRP). Methods A total of 36 ml peripheral venous blood was obtained from 10 healthy adult volunteers, and the PRP was made by secondary centrifugation. The platelet activator was made by bovine thrombin 1000 U in 1 ml 10％ calcium chloride solution. The Thrombin-PRP group was made by PRP and the activator in a ratio of 10:1.The Calcium chloride-PRP group was made in a ratio of 10:1 by PRP and 10％calcium chloride solution instead. The fresh whole blood(whole blood group) and inactived PRP(PRP group) were used as the control groups. The 4 groups were incubated in warm water of 37℃for 0, 1, 8, 24, 72 and 168 h. A quantitative sandwich enzyme-linked immunosorbent assays(ELISA) was used to examine the amount of TGF-β1 and PDGF-AB in different time points of each group. The release curves of TGF-β1 and PDGF-AB were based on afore-mentioned data, and then comparisons of the release curves of TGF-β1 and PDGF-AB in different groups were performed by repeated measurement variance analysis. Results (1)The levels of TGF-β1 and PDGF-AB in the whole blood group and the PRP group continued to increase within 168 h. PRP immediately formed into a gel after mixture with thrombin combined and calcium chloride, and the concentrations of TGF-β1 and PDGF-AB reached the peak&nbsp;in 1 h after activation;increased from (42±21)ng/ml and (77±18)ng/ml to (84±21)ng/ml and (124±35)ng/ml, respectively, and then decreased gradually. The release curve was direct and rapid. The PRP became a gel state in approximate 1 h after mixture with calcium chloride, and the concentrations of TGF-β1 and PDGF-AB were slowly rising and remained high at 168 h. (2)The AUC0-168h of TGF-β1 and PDGF-AB in the PRP group was higher than that in the whole blood group(all P<0.05), and the AUC0-168h of TGF-β1 in the Calcium chloride-PRP group was higher than that in the Thrombin-PRP group(Z=-2.26,P<0.05).However, there was no significant difference in the AUC0-168h of PDGF-AB between the Calcium chloride-PRP group and the Thrombin-PRP group(Z=-1.512,P=0.131). Conclusion Using calcium chloride as activator can get a higher release concentration of TGF-β1 and PDGF-AB and a longer release time, with the largest area under the curve.