体外培养bFGF,PDGF,TGF-β对兔MSC的ALP活性和增殖的影响  

In Vitro Effect of bFGF,PDGF,TGF-β on ALP Activity and Proliferation of Marrow Stromal Cells in Rabbit

作　　者：刘丹平[1] 宋耀滨[1] 关振家[1] 胡蕴玉[2] 

机构地区：[1]锦州医学院附属第一医院骨科,辽宁锦州121000 [2]第四军医大学西京医院全军骨科研究所,陕西西安710032

出　　处：《锦州医学院学报》2003年第1期5-9,共5页Journal of Jinzhou Medical College

摘　　要：目的　在正常生理与病理状态下成纤维细胞生长因子 (FGF)、血小板源性生长因子 (PDGF) ,转化生长因子 β (TGF - β)三种生长因子调节骨的生长发育 ,影响骨折的愈合过程 ,本研究探索在体外培养条件下该三种因子对新西兰白兔骨髓基质细胞增殖与分化的影响 ,为下一步观察这些因子与其它因子联合应用提供基础研究资料。方法　抽取新西兰白兔股骨上穿刺点骨髓进行体外原代培养 ,分离骨髓基质细胞 (Marrowstromalcells,MSC) ,于第 12d行传代培养 ,以 2× 10 3 个细胞 /孔 ,定量接种于 96孔板中 ,同时按照不同剂量加入上述三种因子 ,分别在培养第 10d时用酶动力法检测碱性磷酸酶活性 ,在培养第 5d细胞近于汇合时用MTT法检测细胞的增殖情况。结果　①随着bFGF剂量增加 ,MSCALP活性呈下降趋势 ,当bFGF浓度超过 10 0ng/ml后OD值波动在 0 0 2 0～ 0 0 0 5之间 ,仅是对照组的 1/ 3弱 (P <0 0 1) ,表明MSCALP活性受到了明显的抑制。MTT结果显示 ,OD值随着bFGF的剂量增加而明显上升 ,最高值 (浓度为 6 0 0ng/ml时 )比对照组高出近一倍 ,表明bFGF明显促进MSC增殖 ,有剂量依赖性 (dose -dependent)但当浓度高于 80 0ng/ml时 ,OD值不高于对照组 ,甚至低于对照组 ,表明较高浓度的bFGF抑制MSC增殖 ;Objective To investigate in vitro effects of basic fibroblast growth factor(bFGF),platelet-derivedgrowthfactor(PDGF)andtransforminggrowthfactor-β(TGF-β)onthe differentiation and proliferation of marrow stromal cells(MSC)for obtaining the preliminary data of synergistic effects of these factors and other factors on the MSC as bone tissue enginering cells.Methods To obtain MSC,bone marrow was extracted from superior femoral puncture point of a female New Zealand white rabbit for primary culture for 12 days.Cells were then inoculated at a density of 2×10 3cells/well into 96-well plates and above the three factors at different doses were added to the culture simultaneously and at the time when the medium changed afterwards.The alkaline ghosphatase (ALP)activity of the cells plated were determined using enzyme kinetic method at the 10th day after subculture and the proliferation of the cells were determined with MTT method at the 5th day.Results ①With the increase of the concentration of bFGF,the ALP activity of the MSC was decreasing.While the concentration of bFGF is higher than 100ng/ml,the ALP activity was lower than one third of that of control,demostrating the inhibiting effect of bFGF on the ALP activity of MSC.The results of MTT revealed that the proliferation of the MSC was stimulated by bFGF at dose- dependent concentration,whereas when the concentration is higher than 800ng/ml,the proliferation of MSC was inhibited。②In addition,although the ALP activity and proliferation of the MSC slightly increased as the PDGF concentration increased,no significant difference was observed.③TGF-β appeared to stimulate the ALP activity and proliferation of the MSC,but it was less powerful than rhBMP-2m(unpublished data)in stimulating ALP activity of MSC and less effective than bFGF in promoting proliferation.Conclusion None of the three factors has both the properties of effective promoting proliferation and stimulating ALP activity ,so we should study further to find better combination of the factors re

关 键 词：碱性成纤维细胞生长因子 血小板源性生长因子 转化生长因子β 新西兰白兔 骨髓基质细胞 碱性磷酸活性 增殖 

分 类 号：R329－33[医药卫生—人体解剖和组织胚胎学]