血小板裂解液对人脐带间充质干细胞体外成骨分化的影响

Effect of platelet lysate on osteogenic differentiation of human umbilical cord derived mesenchymal stem cells in vitro

背景:血小板裂解液是浓缩后血小板的裂解产物,含有多种活性成分。有研究表明这些活性成分可促进间充质干细胞的增殖和分化,但血小板裂解液作为一个整体,对间充质干细胞成骨分化的作用尚不明确。目的:分析血小板裂解液对人脐带间充质干细胞成骨诱导分化的干预效应。方法:采用胶原酶消化法分离人脐带间充质干细胞,体外培养扩增,流式细胞仪进行细胞表型鉴定。实验分为以下4组:普通矿化液组,血小板裂解液组,矿化液-血小板裂解液联合诱导组,对照组。加入相应诱导培养基诱导培养2周。结果与结论:分离得到的人脐带间充质干细胞的细胞表型CD34(-)﹑CD45(-)、HLA-DR(-),CD44(+)﹑CD105(+)﹑CD146(+)。茜素红染色在3个诱导组中皆为阳性,染色效果未见明显差异,而对照组阴性。碱性磷酸酶活性测定显示3个诱导组的碱性磷酸酶活性都明显高于对照组(P<0.05),矿化液-血小板裂解液联合诱导组碱性磷酸酶活性明显高于普通矿化液组和血小板裂解液组(P<0.05)。结果显示在体外条件下,单纯的5%血小板裂解液可以诱导人脐带间充质干细胞向成骨细胞定向分化,且联合应用矿化液时能更有效地诱导人脐带间充质干细胞分化为成骨细胞。

BACKGROUND：Platelet lysate is the pyrolysis products of concentrated platelet which contains many active ingredients.Studies have shown that the active ingredients can promote the proliferation and differentiation of mesenchymal stem cells,but the effect of platelet lysate on the osteogenic differentiation of mesenchymal stem cells is not clear.OBJECTIVE：To study the intervention effect of platelet lysate in osteogenic differentiation of human umbilical cord derived mesenchymal stem cells in vitro.METHODS：Human umbilical cord derived mesenchymal stem cells isolated by collagenase digesting were cultured and amplified in vitro.Flow cytometer was used to detect the cell surface markers.The experiment was divided into four groups：mineralization medium group,platelet lysate group,mineralization medium-platelet lysate group and control group.The cells were cultured with corresponding induction medium for 2 weeks,then alizarin red staining was used to identify the oxteoblast,and alkaline phosphatase activity was used to compare the differentiation activity.RESULTS AND CONCLUSION：Flow cytometer results showed that CD34,CD45 and HLA-DR were negative in the human umbilical cord derived mesenchymal stem cells while CD44,CD105 and CD146 were positive.Alizarin red staining was positive in three induced groups with no obvious differences,and it was negative in the control group.The alkaline phosphatase activity level in three induced groups was significantly higher than that in the control group（P 0.05）,and the alkaline phosphatase activity level in mineralization medium-platelet lysate group was significantly higher than that in the mineralization medium group and platelet lysate group（P 0.05）.Results show that 5% platelet lysate alone can induce human umbilical cord derived mesenchymal stem cells to differentiate into osteoblasts in vitro.The induction will be more efficient when combining 5% platelet lysate with mineralization medium.