不同浓度凝血酶对富血小板血浆促骨髓基质细胞增殖影响的研究

The effect of PRP activated by different concentrations of calcium chloride and thrombin on the proliferation of bone marrow stromal cells

目的:探讨不同浓度凝血酶对富血小板血浆(PRP)对骨髓基质细胞(BMSCs)增殖作用的影响。方法:抽取新西兰兔全血制取PRP,按PRP与凝血酶9:1的比例加入20 U/ml4、0 U/ml、60 U/ml、80 U/ml、100 U/ml、500 U/ml和1000 U/ml的氯化钙凝血酶,制取复合生长因子。抽取兔自体骨髓,进行体外培养,取生长良好的第3代细胞消化,以每孔1×104个细胞接种于96孔板,每3孔一组,分别加入含前述生长因子的10%FBS的DMEM、10%FBS的DMEM和无血清DMEM,共9组,用MTT法检测OD值。结果:实验组PRP促BMSCs增殖作用显著高于对照组;实验组中60 U/ml组促增殖作用明显高于其他各组,且20 U/ml^60 U/ml促增殖作用呈浓度依赖关系,高于80 U/ml组作用开始减弱,但80 U/ml^1000 U/ml各组间差别无显著意义。结论:不同浓度凝血酶激活后的PRP均能促进BMSCs的增殖,60 U/ml为最佳浓度。

Objective：This study is to evaluate the effect of platelet-rich plasma （PRP）, activated by different concentrationsof calcium chloride and thrombin, on the proliferation of bone marrow stromal cells （BMSCs）. Method： Platelet-rich plasma （PRP） was obtained from a New Zealand rabbit. The PRP was divided into 7 groups, added in different concentrations of calcium chloride and thrombin at the following concentrations： 20 U/ml, 40 U/ml, 60 U/ml, 80 U/ml, 100 U/ml, 500 U/ml and 1000 U/ml, in proportion as lml thrombin to 9 ml PRP, so that the autologous multiple growth faerors were released from PRP. BMSCs were isolated from the same rabbit, and then culcured in vitro. 1 × 10^4 cells/well were incubated in a 96-well plate at the third passage. 3 wells/group were added in DMEM contained the prepared growth factors and 10% FBS, DMEM contained 10% FBS and DMEM alone. Cell proliferation was assessed utilizing MTT assay. Result：Cell proliferation was enhanced by all PRP from the experimental groups. PRP, activated by calcium chloride and thrombin at the dose of 20 U/ml, 40 U/ml, 60 U/ml, promoted BMSCs growth in a dose-dependant manner. Conclusion： PRP, activated by the different concentrations of calcium chloride bovine thrombin, can promote the BMSCs growth, and 60 U/ml was the best dose.