富血小板血浆对兔骨髓间充质干细胞增殖的作用

Effect of platelet-rich plasma on proliferation of bone mesenchymal stem cells in rabbits

背景:自体富血小板血浆含有多种生长因子,可以克服外源性生长因子成分单一、来源有限、价格昂贵且存在一定的免疫排斥反应等不足。目的:观察富血小板血浆对体外培养骨髓间充质干细胞增殖的影响。设计、时间及地点:区组设计,开放实验,于2004-09/2005-02在同济医学院医学实验公共实验室完成。材料:2月龄新西兰大耳白兔。方法:采用密度梯度离心结合贴壁筛选及传代方法提取、纯化骨髓间充质干细胞,取较纯和生长状态较好的第3代细胞。采用二次离心法提取兔富血小板血浆。在完全培养基加入体积分数为0.2,0.1,0.05富血小板血浆培养骨髓间充质干细胞,以未加入富血小板血浆的完全培养基为对照。主要观察指标:①骨髓间充质干细胞生长曲线。②以MTT比色法观察骨髓间充质干细胞的存活和增殖能力。结果:①加入富血小板血浆干预第2天,骨髓间充质干细胞出现较高的增殖活性,呈快速生长,曲线上升幅度大,细胞倍增时间最短,约30h。以体积分数为0.2富血小板血浆组明显。对照组细胞从第3天起呈快速生长,细胞倍增时间最长,约46h。②富血小板血浆体外培养骨髓间充质干细胞时间越长作用越明显,在培养第3天细胞生长的剂量依赖不明显,培养第6天后较高剂量的富血小板血浆对细胞的增殖作用显著增强,与对照组比较差异有显著性意义(P<0.05,P<0.01)。结论:富血小板血浆能明显促进体外培养骨髓间充质干细胞增殖,并使细胞产生剂量依赖性。

BACKGROUND： Autologous platelet-fich plasma （PRP） containing many growth factors, is superior to exogenous growth factors, which are unsatisfactory due to single component, limited sources, high cost and immunological rejection. OBJECTIVE： To observe the effect of PRP on proliferation of bone mesenchymal stem cells （BMSCs） cultured in vitro. DESIGN, TIME AND SETTING： An open experiment according to block design was performed at the Laboratory of Public Medicine in Tongji Medical College from September 2004 to February 2005. MATERIALS： New Zealand white rabbits aged 2 months were adopted. METHODS： BMSCs were isolated and purified by density gradient centrifugation combined with attachment culture method and transfer of culture. PRP was extracted by the two-step centrifugation method. BMSCs of the third generation were cultured in the complete media containing different concentrations of PRP （0.2, 0.1 and 0.05 volume fractions）, while those cultured in the media without PRP were served as controls. MAIN OUTCOME MEASURES： （1）Growth curves of BMSCs.（2）Survival and proliferation of BMSCs were detected by methyl thiazolyl tetrazolium method. RESULTS： BMSCs proliferation of PRP interfering groups was promoted at day 2, the growth curve increased remarkably, and the capacity of multiplication was stronger. The effect in 0.2 volume fraction of PRP group was the most outstanding and the double time was about 30 hours while that of control group was about 46 hours. Rapid growth emerged in the control group at day 3. PRP contributed to the BMSC cultured in vitro, and the effect was more obvious with the prolonged time. The effect dose-dependence of PRP was not obvious at day 3, but significantly enhanced at day 6 compared with the control group （P 〈 0.05, 0.01）. CONCLUSION： PRP can obviously promote BMSCs to proliferate in vitro, and the effect represents a dose dependence.