背景:已有研究表明富血小板血浆、骨碎补活性成分柚皮甙均可促进间充质干细胞增殖及向成骨分化,二者联合应用的效果尚不明确.目的:观察富血小板血浆联合柚皮甙对人骨髓间充质干细胞体外成骨分化的影响.方法:将第 3 代成人骨髓间充质干细胞分为4组:①空白对照组:α-MEM培养液培养,不加入药物;②富血小板血浆组:加入含富血小板血浆的α-MEM培养液培养;③柚皮甙组:加入含柚皮甙的α-MEM培养液培养;④联合组:加入含富血小板血浆及柚皮甙的α-MEM培养液培养.其中富血小板血浆和柚皮甙含量分别为12.5%、50 μg/L.MTT法检测骨髓间充质干细胞的增殖情况,碱性磷酸酶染色、Ⅰ型胶原免疫组化染色、茜素红染色分析骨髓间充质干细胞成骨分化情况,RT-PCR 检测骨髓间充质干细胞成骨分化过程中相关因子的基因表达.结果与结论:①富血小板血浆、柚皮甙、富血小板血浆+柚皮甙均能促进人骨髓间充质干细胞增殖,其中富血小板血浆+柚皮甙促细胞增殖作用最显著;②碱性磷酸酶染色、Ⅰ型胶原免疫组化染色、茜素红染色均显示富血小板血浆组、柚皮甙组、联合组呈阳性反应,其中联合组阳性反应更明显,空白对照组呈阴性或弱阳性反应;③第7,14天时,富血小板血浆组、柚皮甙组、联合组碱性磷酸酶、Ⅰ型胶原的表达量明显高于空白对照组(P < 0.05),第14天时,联合组碱性磷酸酶、Ⅰ型胶原的表达量明显高于富血小板血浆组、柚皮甙组(P <0.05);④结果表明,富血小板血浆联合柚皮甙对骨髓间充质干细胞体外增殖有促进作用,并能诱导向成骨细胞分化,两者具有相互协同作用.%BACKGROUND: Platelet-rich plasma (PRP) and naringin can both promote proliferation and induce osteogenic differentiation of mesenchymal stem cells. However, their combined use is rarely reported. OBJECTIVE: To observe the effect of PRP combined with naringin on the osteogenic differentiation of human bone marrow mesenchymal stem cells(hBMSCs)in vitro. METHODS: BMSCs at passage 3 were divided into four groups: (1) blank control group, cells were cultured in α-MEM; (2) PRP group, cells were cultured in α-MEM containing PRP; (3) naringin group, cells were cultured in α-MEM containing naringin; and (4) combined group, cells were cultured in α-MEM containing PRP and naringin. The contents of used PRP and naringin were 12.5% and 50 μg/L respectively. Cell proliferation was detected by MTT assay. Expression of related genes in hBMSCs was detected by RT-PCR. Alkaline phosphatase staining, collagen type I immunohistochemical staining, and alizarin red staining were used to analyze the osteogenic differentiation of hBMSCs. RESULTS AND CONCLUSION: The proliferation of hBMSCs was increased in each group, especially in the combined group. Cells in all the groups except the blank control group were positive for alkaline phosphatase staining, collagen type I immunohistochemical staining, and alizarin red staining, and the positive effect was more obvious in the combined group. However, negative or weakly positive response was found in the blank control group. At 7 and 14 days, the expression of alkaline phosphatase and collagen type I was significantly higher in the PRP, naringin and combined groups than the blank control group (P < 0.05); at 14 days, the expression of alkaline phosphatase and collagen type I was significantly higher in the combined group than the PRP and naringin groups (P < 0.05). To conclude, PRP combined with naringin can promote the proliferation of hBMSCs and induce the osteogenic differentiation of hBMSCs. Moreover, there is a synergistic effect between PRP and naringin.
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