富血小板血浆联合钙化诱导对骨髓间充质干细胞增殖和成骨活性的影响

目的:观察富血小板血浆与地塞米松、维生素C、β-甘油磷酸钠联合应用对体外培养的MSCs增殖和诱导成骨的影响。方法:应用密度梯度离心联合贴壁筛选法从兔髂骨骨髓中分离、纯化骨髓间充质干细胞(MSCs),取第3代MSCs随机分为3组,空白对照组加入含10%血清DMEM培养基,钙化诱导组利用含有钙化诱导剂0.1μmol/L的地塞米松、50mg/L的维生素C、10mmol/Lβ-甘油磷酸钠的DMEM培养基,联合诱导组加入含有1%PRP及钙化诱导剂的DMEM培养基,诱导培养8d。结果:MSCs传至第3代细胞多呈长梭多角形。诱导培养5d后,空白对照组碱性磷酸酶染色弱阳性,钙化诱导组碱性磷酸酶染色为阳性,联合诱导组碱性磷酸酶染色为强阳性。诱导培养21d,两诱导组钙结节茜素红染色均呈阳性,MTT法显示诱导培养第2～5d联合诱导组细胞增殖明显高于钙化诱导组(P0.05)。碱性磷酸酶定量测定显示,联合诱导组于第3d、5d和7d,OD值均高于钙化诱导组(P0.05)。结论:富血小板血浆(PRP)与地塞米松、维生素C、β-甘油磷酸钠联合应用于MSCs的成骨诱导,优于单纯钙化诱导方法,且诱导的成骨细胞具有更好的增殖及成骨活性。

Effect of Platelet-rich Plasma Combined with Calcifying Inductors on Proliferation and Osteogenetic Activity of Bone Mesenchymal Stem Cells

Objective To observe the effect of the combined inductors of platelet-rich plasma（PRP） and vitamin C, dexamethasone and β-sodium glycerophosphate on the proliferation mesenchymal stem cells of MSCs and osteoblastic induction in vitro.Methods The MSCs were cultured by density gradient centrifugation and adherence screening from rabbit flank bone.The 3rd MSCs was randomly divided into 3 groups.In the blank group the DMEM medium was used including 10% FCS.In calcifying control group the calcifying DMEM mediuim was used including 0.1 μmol/L dexamethasone, 50 mg/L vitamin C and 10 mmol/Lβ-sodium glycerophosphate.In the combined inductors group the DMEM medium was used including 1% PRP and calcifying inductors, by 8 days of induction.Results The 3rd MSCs mainly showed polygon and whirlpool-like growth.After cultured for 5 days, the alkaline phosphatase staining of the black group showed weak positive, the alkaline phosphatase staining of the two other groups showed positive.Cultured 21 days later, the calcium node staining showed positive.The MTT assay showed the cell proliferation of calcifying control group was significantly higher than combined inductors group on the 2nd to 5th days（P 〈0.05）.ALP quantitative assay showed the OD of the combined inductors group were higher than calcifying control group in 3, 5 and 7th d（P〈 0.05）.Conclusion The combined inductors methods using 1% PRP and dexamethasone, vitamin C and β-sodium glycerophosphate is much better than that using calcifying inductors.The proliferation and osteogenetic activity of the induced osteoblast are superior.