血小板凝胶制备方法的体外实验研究  

Study on the method for platelet gel preparation in vitro

作　　者：单桂秋[1,2] 李艳辉[1,2] 张雅妮[1] 刘广亚[1,2] 吕品[1] 周谋[1] 丘勇新[1] 

机构地区：[1]广州军区广州总医院输血科,广东广州510010 [2]广州中医药大学

出　　处：《中国输血杂志》2011年第4期270-274,共5页Chinese Journal of Blood Transfusion

基　　金：广东省科技计划项目(2009B080701092);广州市科技计划项目(10C36091671);国家科技重大专项--新药创制

摘　　要：目的优化血小板凝胶（platelet gel,PG）的制备方法。方法采用CCK-8试剂盒检测不同浓度外源性凝血酶、不同浓度血小板、血小板与激活剂不同比例制备的PG对ECV304细胞增殖的影响;通过细胞迁移试验检测不同浓度外源性凝血酶对ECV304细胞迁移的影响;采用ELISA试剂盒检测不同浓度外源性凝血酶和不同浓度血小板对VEGF释放量的影响。结果 PG促进ECV304细胞增殖和迁移;加入外源性凝血酶PG形成时间为40~60s,而不加外源性凝血酶PG形成时间600~1 200 s,但是不同浓度（0、100、1 000 U/ml）的凝血酶对ECV304细胞增殖和迁移影响甚微;PG促ECV304细胞增殖作用随血小板浓度的增加而增强,血小板浓度处于（1 500~2 500）×109/L时趋于稳定;富血小板血浆（PRP）与激活剂的比例为10∶1时促细胞增殖效果略优于5∶1;PG上清中VEGF含量随着PRP中血小板含量的增加而增加,不同浓度（0、100、1 000U/ml）凝血酶制备的PG上清中VEGF含量的差异,无统计学意义（P〉0.05）。结论常规采集PRP的血小板浓度（1 000×109/L左右）已能保证细胞增殖效果、满足用于临床的PG需求;PG是否添加激活剂凝血酶应根据临床实际情况选择。Objective To search an optimal method for platelet gel（PG） preparation.Methods The influence of PG prepared under different conditions,including concentrations of exogenous thrombin,concentrations of platelets,proportions of platelet and activating agents on ECV304 cell proliferation were detected by Cell Counting Kit-8 assay.The influence of different concentrations of exogenous thrombin on the ECV304 cell immigration was analyzed with inverted microscope.The influence of different concentrations of exogenous thrombin and different proportions of platelet and activating agents on the content of VEGF were detected with ELISA kit.Results Our study showed that PG stimulated ECV304 cell proliferation and migration.The formation time of PG was 40~60 s upon additon of exogenous thrombin,while it was 600~1 200 s when exogenous thrombin was absent.Different concentrations（0、100、1 000 U/ml） of exogenous thrombin had no effects on ECV304 cell proliferation and migration.PG promoted ECV304 cell proliferation in a platelet concentration-dependent manner,and had a stable tendency when the platelet concentrations was 1 500~2 500×109/L.The proliferation effect was better when the proportion of platelet and activating agent was 10∶1 than 5∶1 instead.Different concentrations（0、100、1 000 U/ml） of exogenous thrombin did not affect ECV304 cell proliferation and migration.There was no significant difference using different concentrations of exogenous thrombin to the release of VEGF.The release of VEGF was raised up according to the increased concentration of platelets.Conclusion The concentration（1 000×109/L） of platelet-rich plasma collected by the routine method could ensured the cell proliferation effect and suitable for clinical use.The suitable proportion of platelet and activating agent for clinical use was 10∶1.The addition of exogenous thrombin should match the practical request in clinic.Our data can provide important reference value to the formation of a standardized methods for PG preparati

关 键 词：血小板凝胶 ECV304细胞 增殖 迁移 血管内皮生长因子 

分 类 号：R331.143[医药卫生—人体生理学] R457.1[医药卫生—基础医学]