富血小板血浆对体外培养骨骼肌干细胞增殖及成骨活性的作用

Effects of platelet-rich plasma on proliferative and osteogenetic activity of skeletal muscle stem cells in vitro

背景:已有研究发现富血小板血浆可以影响骨髓间充质干细胞和脂肪干细胞的生长和分化。目的:观察富血小板血浆对体外培养的骨骼肌干细胞增殖与诱导成骨的影响,探讨富血小板血浆影响骨骼肌干细胞的机制。设计、时间及地点:随机对照动物实验,于2008-06/10在上海交通大学附属第六人民医院中心实验室完成。材料:普通级新西兰大白兔9只,体质量2.5～3kg,年龄1岁左右,雌雄不限。方法:取兔右后肢比目鱼肌体外培养新西兰大白兔骨骼肌干细胞。取兔耳中央动脉血,经离心处理后制备富血小板血浆。将细胞分为实验组与对照组,实验组以含12.5%自体富血小板血浆的条件培养液干预,对照组不进行富血小板血浆干预。主要观察指标:①细胞形态学观察。②以四甲基偶氮唑盐法检测细胞增殖活性。③以碱性磷酸酶钙钴法染色、碱性磷酸酶活性测定、茜素红染色和骨钙素免疫荧光染色检测细胞成骨活性。结果:四甲基偶氮唑盐法显示富血小板血浆诱导实验组较对照组增殖明显(P<0.01);碱性磷酸酶活性较对照组增高明显(P<0.01)。富血小板血浆诱导实验组碱性磷酸酶染色阳性,骨钙素免疫荧光染色阳性,茜素红染色可见钙结节形成。结论:富血小板血浆对体外培养的骨骼肌干细胞增殖与成骨分化活性具有显著的促进作用。

BACKGROUND： Studies has confirmed that platelet-rich plasma （PRP） can affect the proliferation and differentiation of bone marrow mesenchymal stem cells and adipose derived stem cells. OBJECTIVE： To observe the effects of PRP on the proliferative and osteogenetic activity of skeletal muscle stem cells （SMSCs） in vitro, in addition, to etucidate the potential mechanism by which PRP affects SMSCs. DESIGN, TIME AND SETTING： A randomized controlled animal experiments was completed in the Center Laboratory of the Sixth People＇s Hospital Affiliated to Shanghai Jiao Tong University from June to October in 2008. MATERIALS： Nine New Zealand, ordinary grade rabbits, weight of 2.5-3.0 kg, 1 year old, irrespective of gender. METHODS： The right soleus was dissected and cultured for SMSCs. PRP was prepared with central artery of rabbits＇ ears. The cells were randomly divided into the experimental group and the control group. In the experimental group, the SMSCs were interfused with PRP （special culture media including 12.5% PRP）. There was no intervention in the control group. MAIN OUTCOME MEASURES： ①Morphological observation. ②The proliferative ability of the ceils was detected with MTT. ③ The osteogenetic ability was measured with alkaline phosphatase （AKP） staining, alizarin red staining and osteocalcin immunofluorescence assay. RESULTS： MTT method showed that the proliferative activity of the experimental group was obviously stronger than that of the control group （P 〈 0.01 ）. There was a significant increase in AKP activity of the experimental group compared to the control group （P 〈 0.01）, The SMSCs in the experimental group was positive to AKP and osteocalcin immunofluorescence assay, and calcium nodules could be seen after alizarin red staining. CONCLUSION： PRP exhibits promotive effect on the proliferation and osteogenic differatiation of SMSCs in vitro.