摘要
目的探讨人血小板裂解液(human platelet lysates,HPL)对骨髓间质干细胞(mesenchymal stem cells,MSCs)增殖以及生物学特性的影响。方法通过对机采血小板反复冻融获得HPL,采用密度梯度离心法从骨髓中分离MSCs,分别用10%胎牛血清(feotal calf serum,FCS)和LD-DMEM添加不同浓度的HPL进行培养,以确定最佳HPL浓度。分离6株人源MSCs,从原代开始分别用添加胎牛血清和最佳HPL浓度的LD-DMEM进行培养,对两种培养体系下6株不同来源的MSCs的增殖能力,表面标记表达量以及细胞周期进行研究,并利用t-test进行统计学分析,P<0.05视为有统计学差异。结果研究发现HPL中含有MSCs生长所必需的4种生长因子,PDGF-AB(人血小板衍生生长因子AB),bFGF(成纤维细胞生长因子),IGF-1(胰岛素样生长因子1),TGF-β1(转化生长因子β1),浓度分别为:(0.53±0.06)ng/ml,(37.5±4.31)pg/ml,(0.15±0.06)mg/ml,(5150±463)pg/ml。适合MSCs增殖的最佳HPL浓度为7.5%。两种培养条件下的细胞形态无明显区别,均为长梭形。8代以前,细胞增殖能力没有显著性差异(P>0.05),两种培养条件下的MSCs均表达CD105,CD106以及粘附分子CD29和CD44,不表达CD34,CD45,且其表达量没有显著性差异(P>0.05)。两种培养体系下绝大多数细胞处于G0-G1期,FCS培养的MSCs其G0-G1期占(96.97±0.95)%,7.5%的HPL培养的MSCs其G0-G1期占(94.58±1.56)%,两种培养条件下不同周期没有显著性差异(P>0.05)。结论本研究适合MSCs增殖的最佳HPL浓度为7.5%,HPL可以替代胎牛血清体外培养扩增人骨髓间质干细胞,并保持其生物学特性基本不变。
Objective To investigate the effect of human platelet lysates（HPL） obtained from platelet-rich plasma on the proliferation and biological characteristics of human mesenchymal stem cells（MSCs） in vitro.Methods HPL was obtained by repeated freeze-thawing of human plateletes,and the MSCs separated by density gradient centrifugation from 6 donors were expanded in medium supplemented with 10% fetal bovine serum（FCS） or HPL at different concentrations.The optimal concentration of HPL for cells culture was determined according to the cell proliferation kinetics.The cultured MSCs were characterized for their proliferation,cell phenotype,and cell cycle distribution.Results The HPL-supplemented medium contained 4 essential growth factors for the growth of MSCs,namely platelet-derived growth factors（0.53±0.06 ng/ml）,basic fibroblast growth factor（37.5±4.31 pg/ml）,insulin-like growth factor-1（0.15±0.06 mg/ml） and transforming growth factor（5150±463 pg/ml）.Cultured in the presence of HPL at the optimal concentration of 7.5%,the MSCs displayed a spindle-shaped fibroblast-like morphology without obvious changes in the proliferation activity till passage 8（P0.05）,similar to those of cells in FCS-supplemented culture medium.Flow cytometry and cell cycle analysis revealed no differences in the phenotypes or cell cycle distribution between the cells cultured in the presence of 7.5% HPL and 10% FCS.Conclusion The culture medium supplemented by 7.5% HPL can promote the expansion of human MSCs and maintain the basic biological characteristics of the cells.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2011年第8期1396-1400,共5页
Journal of Southern Medical University
基金
广州市医药卫生科技项目(2008-Zdi-10)
关键词
骨髓间质干细胞
血小板裂解液
增殖
表面标记
细胞周期
human platelet lysates
mesenchymal stem cells
surface markers
proliferation kinetics
cell cycle
