不同方法制备的富含血小板血浆裂解液对成人骨髓间充质干细胞增殖、成骨的影响

Platelet Rich Plasma Pyrolysis Liquid Prepared with Different Methods on Proliferation and Osteogenesis of Adult Bone Marrow Mesenchymal Stem Cell

目的:用不同方法制备富含血小板血浆(platelet-rich plasma,PRP)裂解液,在不同体积分数下找到最适成人骨髓间充质干细胞(human bone marrow stromal cells,hBMSCs)体外增殖及其向成骨细胞分化的PRP裂解液制备方法。方法:抽取成人骨髓20mL,采用密度梯度离心法分离骨髓,贴壁培养法获得成人骨髓间充质干细胞;抽取静脉血80mL,分别用1次离心法、2次离心法和3次离心法制备PRP,进行血小板计数分析。-80℃冻存后,反复解冻、冻存至少3次以上获得PRP裂解液;将第2代细胞分别培养在体积分数为5%、10%、15%、20%的不同方法获得的PRP裂解液的培养液中,24、48、72、96h后检测细胞的增殖状况;将第2代细胞分别培养在体积分数为5%、10%、15%、20%的PRP裂解液和不加PRP裂解液的成骨细胞诱导液中,观察骨髓间充质干细胞的成骨表达以及PRP裂解液对其成骨矿化的影响。结果:各组PRP裂解液均促进hBMSCs增殖作用,差异有统计学意义(均P<0.01),各组细胞增殖在体积分数15%PRP裂解液组、72h时达到峰值,其中3次离心方法获得的细胞增殖最明显。Vonkossa’s染色观察细胞的成骨作用,各组成骨细胞数量随着PRP裂解液浓度上升而增多。3种方法获得的PRP裂解液都在培养液体积分数15%时获得的成骨细胞数量最多,其中第3种离心方法获得的矿化结节较第1种离心方法、第2种离心方法明显。结论:PRP裂解液对hBMSCs的体外增殖有促进作用,并且第3种离心法制备PRP裂解液对细胞成骨表达和成骨矿化作用最明显。

Objective：To find the most suitable method of platelet-rich plasma （PRP） lysate preparation for proliferation and osteogenesis of human bone marrow stromal cells （BMSCs） in vitro using different methods of PRP preparation. Methods： Human BMSCs were obtained by density gradient centrifugation and adherent culture. PRP was obtained from venous blood by centrifugation for once, twice and three times respectively for platelet count. PRP lysate was obtained by repeat cryopreservation and thawing. Then the second generation BMSCs were cultured respectively in medium with 5%, 10%, 15 %, and 20% PRP lysate. After 24h, 48h, 72h, and 96h, cell proliferation was detected. The second generation BMSCs were cultured in medium with or without 5 ~, 10 ~, 15 ~, and 20 ~ PRP lysate for detection of osteogenic expression and bone mineralization. Results：PRP lysate promoted the proliferation of human BMSCs in all groups （all P^0.05）, with the most obvious effect in 15 % PRP lysate cultured for 72h with three times of centrifugation. Vonkossa＇s staining showed that the osteoblasts increased with higher concentration of PRP lysate. There were more osteoblasts when there was 15 % PRP lysate in the medium. More mineralized nodules were obtained by the three-time centrifugation method than the once and twice centrifugation methods. Conclusion：PRP lysate can promote human BMSCs proliferation in vitro, and PRP lysate prepared with three-time centrifugation has the most obvious effect on osteogenic expression and bone mineralization.