血小板裂解液对大鼠骨髓间充质干细胞生长的影响

Effects of platelet lysate on the proliferation of rat bone marrow mesenchymal stem cells

背景:血小板裂解液是将浓缩血小板进一步裂解后所获得的液体成分,含有多种生物活性因子。有研究认为血小板裂解液可促进成骨细胞的增殖,而其对骨髓间充质干细胞生物学功能的影响还不太了解。目的:探讨血小板裂解液对大鼠骨髓间充质干细胞生长增殖的影响。设计、时间及地点:独立样本观察,于2007-09/2008-01在山西省医用组织库完成实验。材料:清洁级成年健康Wistar大鼠8只。方法:取2只大鼠股骨,全骨髓培养法传代扩增骨髓间充质干细胞。自6只大鼠心内采血,采用3次离心结合反复冻融法制备血小板裂解液。取第5代骨髓间充质干细胞,以含10%胎牛血清的L-DMEM/F12作为基础培养基组,在此基础上分别加入血小板裂解液至终浓度为1%和5%作为条件培养基组。主要观察指标:ELISA法测定血小板裂解液中生长因子含量。CASY细胞分析仪计数活细胞。双缩脲法全自动生化分析仪检测细胞总蛋白含量。倒置显微镜动态观察细胞形态变化与生长状况。结果:①血小板裂解液中的血小板衍生生长因子、转化生长因子β1、胰岛素样生长因子1和血管内皮细胞生长因子含量分别为(300±30),(140±25),(80±35),(70±20)ng/L。②自培养第3天开始,条件培养基组活细胞数均明显高于基础培养基组(P<0.05),且含5%血小板裂解液的条件培养基组升高幅度尤为显著,于第3天达到对数生长期,比基础培养基组提前约1d,细胞总数在第4,9天时达基础培养基组的近3倍。③培养1周后条件培养基组每106个细胞中的总蛋白含量均显著高于基础培养基组(P<0.05)。④骨髓间充质干细胞在不同浓度血小板裂解液培养条件下,细胞形态基本一致,多为长梭形,类似于成纤维细胞。结论:血小板裂解液是多种生长因子的承载体系,具有丝裂原功效,可在体外促进大鼠骨髓间充质干细胞的生长增殖,且该效应存在一定程度的剂量依赖性。

BACKGROUND： Platelet lysate containing various bioactive factors is the fluid obtained from platelet concentrate after further splitting. Some studies have demonstrated that platelet lysate can promote osteoblast proliferation. However, the effect of platelet lysate on the biological function of bone marrow mesenchymal stem cells （BMCs） is still unknown. OBJECTIVE： To explore the effects of platelet lysate on the proliferation of rat BMCs in vitro. DESIGN, TIME AND SETTING： Independent sample observation was performed at the Shanxi Provincial Tissue Bank from September 2007 to January 2008. MATERIALS： Eight clean-grade healthy adult Wistar rats. METHODS： Femora of two rats were selected and BMCs were passaged using whole bone marrow culture method. Platelet lysate was prepared through three times of centrifugations and repeated freeze-thaw for the venous blood aspirated from cardiac cavities of 6 rats. The 5^th passage of cells were cultured in L-DMEM/F12 medium containing 10% fetal bovine serum as basic culture group, and cells cultured in platelet lysate （terminal 1% and 5%） based on the basic culture medium were regarded as conditioned culture groups. MAIN OUTCOME MEASURES： ELISA assay was conducted to detect the concentration of growth factors in platelet lysate. Cell counting was performed by CASY cell analyzer. Total protein content in three groups was determined by Biuret method. The growth and morphology of cells were observed by inverted phase contrast microscope. RESULTS： ELISA assay showed that the content of platelet-derived growth factor, transforming growth factor- β 1, insulin-like growth factor-1 and vascular endothelial cell growth factor in platelet lysate attained to （300±30）, （140±25）, （80±35）, （70±20） ng/L, respectively. Since the 3^rd day, conditioned culture groups showed markedly high rate of proliferation than basic culture group （P 〈 0.05）, especially 5% group, which displayed about 3 folds of number of viable cells on days of 4 and 9 （P 〈 0.05）. The total protein content in each 10^6 cells of conditioned culture groups was significantly higher than that of the basic culture group （P 〈 0.05）. Cells cultured in platelet lysate displayed similar configuration with long shuttle-shape as fibroblasts. CONCLUSION： Platelet lysate is a kind of system carrying various growth factors and functions as a kind of mitogen of BMCs in rats in a dose-dependent manner.