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目的探讨血小板裂解液(platelet lysate,PL)在体外定向诱导人脐带间充质干细胞(human umbilical cord derived mesenchymal stem cells,hUCMSCs)分化成软骨细胞中的作用。方法取健康产妇自愿捐赠脐带,采用胶原酶消化法分离hUCMSCs,体外培养扩增,流式细胞仪进行细胞表型鉴定。根据加入诱导培养基成分不同将实验分为以下3组:A组为H-DMEM培养基、10%FBS及10%PL,B组为H-DMEM培养基、10%FBS、10 ng/mL TGF-β1、1×10-7 mol/L地塞米松、50μg/mL维生素C及1%胰岛素铁硒传递蛋白(insulin-transferrin-selenium,ITS),C组为H-DMEM培养基、10%FBS、10 ng/mL TGF-β1、1×10-7 mol/L地塞米松、50μg/mL维生素C、1%ITS及10%PL。诱导培养2周,甲苯胺蓝染色检测各组软骨细胞基质的分泌,免疫荧光检测软骨特异性Ⅱ型胶原表达,半定量RT-PCR检测蛋白聚糖(Aggrecan)和Ⅱ型胶原表达。结果分离得到的hUCMSCs不表达造血细胞的表面标记CD45、CD34和HLA-DR,而表达黏附分子和MSCs表面标记CD44、CD105和CD146。甲苯胺蓝染色和Ⅱ型胶原免疫荧光染色示C组呈阳性,B组呈弱阳性,而A组均呈阴性。半定量RT-PCR检测示Aggrecan和Ⅱ型胶原在B、C组中均有表达,A组中未见表达;C组Aggrecan mRNA和Ⅱ型胶原mRNA表达明显高于B组,差异均有统计学意义(P<0.05)。结论单纯10%PL不能诱导hUCMSCs成软骨分化,但它可当作成软骨诱导培养基的辅助添加剂,对hUCMSCs成软骨分化有明显促进作用,为构建组织工程软骨提供了新的可利用条件。
Objective To investigate the role of platelet lysate (PL) in differentiating into human umbilical cord derived mesenchymal stem cells (hUCMSCs) into chondrocytes in vitro. Methods The umbilical cord was donated voluntarily by healthy mothers. The hUCMSCs were isolated by collagenase digestion and cultured in vitro. The cell phenotypes were identified by flow cytometry. The experiments were divided into three groups according to the different inducing medium components: group A was H-DMEM medium, 10% FBS and 10% PL, group B was H-DMEM medium, 10% FBS and 10 ng / mL TGF β1, 1 × 10-7 mol / L dexamethasone, 50μg / mL vitamin C and 1% insulin-transferrin-selenium (ITS) , 10 ng / mL TGF-β1, 1 × 10-7 mol / L dexamethasone, 50 μg / mL vitamin C, 1% ITS and 10% PL. After induction for 2 weeks, the secretion of chondrocytes was detected by toluidine blue staining. The expression of cartilage-specific type Ⅱ collagen was detected by immunofluorescence. The expression of aggrecan and type Ⅱ collagen was detected by semi-quantitative RT-PCR. Results The isolated hUCMSCs did not express the surface markers CD45, CD34 and HLA-DR of hematopoietic cells, but expressed CD44, CD105 and CD146 on the surface of adherent cells and MSCs. Toluidine blue staining and type Ⅱ collagen immunofluorescence staining showed that C group was positive, B group was weakly positive, while A group was negative. Semi-quantitative RT-PCR showed that Aggrecan and type II collagen were expressed in B and C groups but not in A group. Aggrecan mRNA and type II collagen mRNA expression in group C were significantly higher than those in group B Significance (P <0.05). Conclusions 10% PL alone can not induce hUCMSCs to differentiate into cartilage, but it can be used as an auxiliary additive in cartilage induction medium, which can promote hUCMSCs to differentiate into cartilage and provide new conditions for the construction of tissue engineered cartilage.