摘要
目的探讨在不同冻融条件下血小板裂解液(PL)制备的最适条件。方法由南部战区血液中心提供50人份PRP(30mL/人份),每份PRP再随机分为3等分,分别为A、B、C 3组。将A、B两组PRP分别至于-20℃(A组)、-80℃(B组)冰箱保存24 h后,反复冻存融化6次,获取血小板能裂解液;C组添加牛凝血酶和钙离子,激活剂与PRP按照1∶9的比例添加获取PRP上清液。应用酶联免疫吸附试验法定量检测A、B、C 3组中血小板源性生长因子(PDGF-AA)、转化生长因子β(TGF-β)、胰岛素样生长因子(IGF)的浓度;细胞增殖实验采用CCK-8试剂检测在A、B、C 3个实验组制备的上清液与对照组在24 h、48 h、72 h对小鼠软骨细胞增殖的影响。结果生长因子含量检测:A、B、C3组中PDGF-AA的含量分别为234.75±12.73、236.58±32.64、244.25±23.29(P>0.05);TGF-β的含量分别为589.66±53.54、575.42±70.96、518.08±111.7(P>0.05);IGF的含量分别为15.77±1.65、16.42±0.88、14.24±1.67(P>0.05);3组间3种生长因子含量均无统计学差异。小鼠软骨细胞增殖实验(OD值):实验A、B、C 3组与对照组在小鼠软骨细胞培养24h时,分别为:0.06±0.02、0.05±0.02、0.05±0.01、0.01±0.01;48 h时分别为0.22±0.13、0.11±0.03、0.24±0.12、0.04±0.02;72h时分别为0.32±0.08、0.28±0.12、0.27±0.11、0.08±0.03;其中实验组3组在24、48、72 h时均优于对照组(P<0.05),A、B、C3组组内比较,24、48、72h时差异均无统计学意义(P>0.05)。结论-20℃冻融温度制备的PL在某些治疗上可以代替牛凝血酶激活上清液应用于临床,而且更加安全。
Objective To explore the optimum conditions for the preparation of platelet lysate(PL) under different freeze-thaw conditions. Methods 50 copies of platelet rich plasma(PRP) were provided by Southern theater blood center. Each PRP is randomly divided into 3 equal parts, namely, A, B and C three groups. PRP in groups A and B were stored in the refrigerator at-20℃(group A) and-80℃(group B) for 24 hours, and then repeatedly frozen and thawed 6 times to obtain platelet lysate. Group C was added with bovine thrombin and calcium ions, and activator and PRP were added in a ratio of 1:9 to obtain PRP supernatant. The concentrations of platelet-derived growth factor(PDGF-AA), transforming growth factor beta(TGF-β), and insulin-like growth factor(IGF) in groups A, B, and C were detected by enzyme-linked immunosorbent assay. Cell proliferation assay was performed using CCK-8 reagent to detect the effects of supernatants prepared from three experimental groups A, B, and C on the proliferation of mouse chondrocytes at 24, 48, and 72 h. Results Detection of growth factor content: the contents of PDGF-AA in the three groups A, B and C were 234.75±12.73, 236.58±32.64, 244.25±23.29 respectively(P>0.05);the contents of TGF-beta in group A, B and C were 589.66±53.54), 575.42±70.96) and 518.08±111.7) respectively((P>0.05);The contents of IGF in group A, B and C were 15.77±1.65, 16.42±0.88, 14.24±1.67 respectively(P>0.05). There was no significant difference in the contents of three growth factors between the three groups. Mouse chondrocyte proliferation test(OD value): The OD values of experimental group A, B, C and control group were 0.06±0.02、0.05±0.02、0.05±0.01、0.01±0.01 at 24 hours, 0.22±0.13、0.11±0.03、0.24±0.12、0.04±0.02 at 48 hours, and 0.32±0.08、0.28±0.12、0.27±0.11、0.08±0.03 at 72 hours, respectively. The experimental groups were better than the control group at 24, 48 and 72 hours(P< 0.05). There was no significant difference among groups A, B and C at 24, 48 and 72 hours(P > 0.05). Conclusion PL prepared at-20 C freeze-thaw temperature can replace bovine thrombin activation supernatant in some treatments, and safer.
作者
刘广亚
许育兵
张喻
朱展鸿
单桂秋
LIU Guangya;XU Yubing;ZHANG Yu;ZHU Zhanhong;SHAN Guiqiu(Department of Blood Trafiision, General Hospital of Southern Theater Command,Guangzhou 510010, China)
出处
《中国输血杂志》
CAS
2019年第6期545-548,共4页
Chinese Journal of Blood Transfusion
基金
军队后勤科研计划项目(BGZ15C002)
广东省医学科学技术研究基金(A2018221)
关键词
温度
血小板裂解液
生长因子
软骨细胞
细胞增殖
temperature
platelet lysate
growth factor
chondrocytes
cell proliferation
