contrast microscope. Identification of cell surface antigen by flow cytometry showed that CD29 and CD90 expressed positively and CD34, CD45 expressed negatively in the 2nd passage of rBMSCs. Adipogenic and osteogenic differentiation a-bility of the cultured rBMSCs were confirmed after induction. MTT assay showed that at 1, 3, 5, 7 and 9 days, cell proliferation was significantly higher in the PRP group than that of control. ALP assay showed that after 7 d and 14 d, cells were cultured by ostoegenic induction medium, ALP activity was lower than ostoegenic induction medium and PRP group. The ALP activity was higher on 14d than on 7d(P<0.05). Gene expressions of Col-3 and OPN in the cells stimulated by 10% PRP was slightly lower than the cells stimulated by ostoegenic induction medium, but higher than the cells stimulated by a-MEM medium only. Col-3 and OPN expression in the cells stimulated by both ostoegenic induction medium and PRP were much higher than other groups (P < 0. 05). The results of SEM showed promoted cell attachment and stretching area under 10% PRP stimulation. CONCLUSION: PRP can promote proliferation, osteogenic differentiation, collagen synthesis of the rBMSCs, as well as their attachment capability to the biomaterials.%目的:探讨富血小板血浆(platelet - rich plasma,PRP)对大鼠骨髓间充质干细胞(rat bone marrow mesenchymal stem cells,rBMSC)体外成骨能力的影响.方法:体外分离、培养rBMSC,经细胞表面标记物检测和体外诱导分化鉴定后,分别采用WTT法检测PRP对细胞增殖活性的影响；细胞化学法检测PRP对成骨诱导后rBMSC碱性磷酸酶(alkaline phosphate,ALP)活性的影响；RealTime- PCR检测PRP对细胞成骨分化指标Col-3、OPN mRNA表达水平的影响；扫描电镜观察PRP对rBMSC在Bio-Oss上粘附的影响.结果:rBMSC 传代后细胞生长状态相对稳定,呈梭形；细胞表面标记物CD29阳性率为88.2％,CD90阳性率为98％,CD34细胞阴性率为2.8％,CD45细胞计数阴性率为2.7％；成骨诱导21 d后,茜素红染色可见明显矿化结节；成脂诱导14d后,油红O染色可见红色脂滴形成.MTT检测,10％ PRP组在培养1、3、5、7、9d各时间点的OD值均显著高于空白对照组(P＜0.05).ALP染色显示,无论是诱导培养7d还是14 d,成骨诱导液+PRP组的ALP活性均明显高于单纯成骨诱导液组,差异有统计学意义(P＜0.05)；另外,各组不同诱导培养时间点相比还发现,无论是实验组还是对照组,在诱导7d时,ALP活性均较低；而在14 d,ALP的活性明显升高.RealTime-PCR检测显示,单纯10％ PRP诱导培养7d和14 d后细胞Col-3、OPN mRNA水平虽稍低于成骨诱导液组,但明显高于空白对照组,而10％ PRP联合成骨诱导液诱导培养7d和14d的Col-3、OPN mRNA的表达量更高,与其他各组相比差异均有统计学意义(P＜0.05).另外各组诱导培养14d的Col-3、OPN mRNA水平显著高于相应的7d组(P＜0.05).扫描电镜结果显示PRP明显促进rBMSC在Bio-Oss骨粉上的粘附与伸展.结论:PRP对rBMSC的增殖、成骨分化,以及在生物材料上的粘附能力均具有明显的促进作用.
展开▼
