富血小板血浆对体外培养骨髓间充质干细胞增殖及成骨活性的作用

EFFECTS OF PLATELET-RICH PLASMA ON PROLIFERATION AND OSTEOGENETIC ACTIVITY OF MARROW MESENCHYMAL STEM CELLS IN VITRO

目的 观察富血小板血浆 (platelet- rich plasma,PRP)在体外培养骨髓间充质干细胞 (marrowmesenchymal stem cells,MSCs)的增殖及成骨作用 ,探讨 PRP促进骨修复的细胞和分子机制。 方法 体外培养人MSCs,分为实验组 (n=9)与对照组 (n=9) ,实验组进行 PRP干预 (10μl/ ml培养液 )。于不同时间点收集两组细胞 ,以流式细胞仪检测 S期比例 ,MTT法检测细胞增殖活性 ,碱性磷酸酶 (alkaline phosphatase,AL P)测定和四环素荧光法检测细胞成骨活性 ,逆转录 PCR检测成骨启动基因 cbfa1的表达。 结果 PRP作用于 MSCs,可刺激细胞增殖 ,流式细胞仪检测 PRP加入后 2 4 h S期比例由对照组 7.2± 0 .5至 14 .5± 0 .4具有统计学意义 (P<0 .0 1) ,MTT法显示 PRP作用后细胞增殖加速 ,第 4天达到平台期。而 AL P活性在作用后 3、6和 9d达到 7.79± 1.98、9.5 1± 2 .31和 14 .0 3± 3.0 2 ,与对照组 2 .0 6± 0 .77、2 .84± 0 .82和 2 .5 8± 0 .84组间比较差异有统计学意义 (P<0 .0 5 )。矿化结节形成增多 ,逆转录 PCR显示 cbfa1的表达在 PRP加入 2、4和 8h逐渐加强。 结论 PRP对骨修复的促进作用与其促进 MSCs增殖、加强成骨启动基因的表达、增强成骨活性的作用有关。

ObjectiveTo study the effects of plateletrich plasma(P RP ) on the proliferation and osteogenetic activity of the marrow mesenchymal stem cells(MSCs) cultured in vitro to elucidate the cellular and molecular mechanism by which PRP accelerates bone repair.〓Methods〓The human MSCs w ere cultured in vitro and randomly divided into the experimental group(?n=9)? and control group(?n=9)?. In the experimental group, the MSCs were interfused with PRP(10 μl/ml culture media). The proliferation ability of the cells was te sted by flow cytometry and MTT, the osteogenetic activity by alkaline phosphatas e(ALP) measuring and tetracycline gluorometry, and cbfal mRNA expression by reve rse transcriptPCR.Results PRP could stimulate the MSCs prol iferation. The flow cytometry assay showed that the MSCs proportion of S period of the experimental group significantly increased 14.5±0.4 in comparison with t hat of the control group 7.2±0.5 (?P￡1/40.01)? after 24 hours. MTT value showed that MSCs proliferated to platform period earlier in the experimental group tha n in the control group. There was a significant increase in ALP activity of the experimental group 7a±79?à1a±98,a(c)951±231 and 14.03±3.02 when compared w ith that of the control group 2.06±0.77,2.84±0.82 and 2.58±0.84 after 3, 6 an d 9 days(?P￡1/40.05)?. The number of mineral nodes increased. Reverse transcript PCR showed that the expression of cbfal mRNA were elevated gradually at 2,4 an d 8 hours after interfused with PRP.Conclusion The effect of PRP on accelerating bone repair is related to its effects on stimulating the pro liferation of MSCs, increasing the cbfal expression and promoting the osteogenet ic activity.