富血小板纤维蛋白对兔骨髓间充质干细胞成软骨分化的影响

Effect of Platelet-rich Fibrin on Chondrogenic Differentiation of Rabbit Bone Marrow Mesenchymal Stem Cells

目的:观察自体富血小板纤维蛋白(platelet-rich fibrin,PRF)对体外培养的兔骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)成软骨分化的影响。方法:兔心脏采血制备PRF,电镜观察其超微结构;分离培养兔BMSCs,取第3代细胞用于实验,分为PRF组、阳性对照组、空白对照组。诱导培养21d后,对三组细胞分别进行形态学观察,成软骨鉴定染色(甲苯胺蓝、II型胶原免疫组化染色),软骨相关基因表达检测(Ⅱ型胶原、Aggrecan、SOX9)。结果:PRF组和阳性对照组中BMSCs经诱导后,细胞由长梭形变为三角形、多角形、圆形;甲苯胺蓝、II型胶原免疫组化染色均为阳性;Ⅱ型胶原、Aggrecan、SOX9基因表达水平均较高,两组比较无统计学差异,空白对照组未见相关分化现象。结论:PRF在体外可促进兔BMSCs成软骨分化,可作为自体生物材料,在构建组织工程软骨中发挥更好的作用。

Objective：To investigate the effect of autologous platelet rich fibrin （PRF） on chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells （BMSCs） in vitro. Methods： Blood extracted from rabbit＇s heart was used to prepare PRF, and the ultrastructure of PRF was observed by electron microscope. BMSCs were isolated and cultured ex rive. BMSCs at passage 3 were divided into three groups： PRF group, positive control group and blank control group. After 2 weeks of culture, the morphology of cells in each group were observed by phase-contrast microscope, the phenotype was identified with toluidine blue staining and collagen Ⅱ immunocytochemistry, and the gene expression levels of collagen type Ⅱ, Aggrecan, SOX 9 were detected by Real-time PCR. Results： Phase-contrast microscope showed that inducing BMSCs of PRF group and positive control group grew form long spindle to triangle, polygon, and circle. The toluidine blue staining and collagen Ⅱ immunocytochemistry of two groups were positive, gene expression levels of collagen type Ⅱ, Aggrecan, and SOX 9 were high, there was no statistical difference between two groups. However, cells in the control group were not detected these changes. Conclusion： PRF could promote chondrogenic differentiation of BMSCs ha vitro. It can be used as autologous biological materials, and it will play an important role in constructing tissue engineering cartilage.