富血小板血浆对人脂肪间充质干细胞增殖及成骨分化能力的影响

Effect of Platelet-rich Plasma on the Proliferation and Osteogenic Differentiation of Human Adipose-derived Mesenchymal Stem Cells in Vitro

目的:探讨富血小板血浆(PRP)对人脂肪间充质干细胞(hADSCs)增殖、分化及成骨能力的影响。方法:脂肪组织体外分离获得hADSCs,2次离心制备PRP,氯化钙加人凝血酶激活PRP。倒置相差显微镜观察成骨诱导实验中PRP对细胞增殖、分化状况的影响,钙-钴法检测PRP作用下hADSCs矿化结节形成,碱性磷酸酶(ALP)检测PRP对细胞活性及分化能力的影响。CCK-8法检测成骨诱导组和非成骨诱导组加PRP培养后2、4、6、8、16d细胞活性变化。结果:成骨诱导实验中50%PRP30μL组细胞密集、数量最多,且有剂量依赖性;钙-钴法检测见PRP原液组细胞染色最深,且有浓度依赖性;碱性磷酸酶显色见PRP原液组细胞活性最强,染色最深。CCK-8检测显示无论有无成骨诱导,各浓度PRP均可促进细胞增殖,且有浓度依赖性。结论:适宜浓度的PRP可明显促进hADSCs增殖,并有浓度及剂量依赖性。

Objective： To evaluate the effect of platelet-rich plasma（PRP） on proliferation and osteogenic differentiation of human adipose-derived mesenchymal stem cells（hADSCs） in vitro.Methods： hADSCs were separated from an adult donor.PRP was prepared by twice centrifugation method.CaCl2 combined with thrombin was used to activate PRP.In the osteogenesis induction experiment the morphology of cells was studied with a phase contrast microscope.The alkaline phosphatase（ALP） activity of hADSCs in different concentrations PRP groups was determined by calcium-cobalt method and BCIP/NBT alkaline phosphatase color development kit was used,respectively.The proliferation of hADSCs with and without osteogenic induction culture medium in various concentrations PRP groups was detected by CCK-8 assay in 2nd,4th,6th,8th and 16th day.Results： The microscope investigation showed that 50% PRP 30μL group improved cell proliferation higher than other groups.The significant increase in ALP was observed when added original liquid PRP.The CCK-8 showed that mean A450 of original liquid PRP was higher than other groups whichever with or without osteogenic induction.Conclusion： These data suggest that PRP has a positive dose-depend and concentration-depend influence on hADSCs proliferation.