血小板裂解液对人脐带间充质干细胞体外成软骨分化的影响  

EFFECT OF PLATELET LYSATE ON CHONDROGENIC DIFFERENTIATION OF HUMAN UMBILICAL CORD DERIVED MESENCHYMAL STEM CELLS IN VITRO

作　　者：冯学涛[1] 田少奇[1] 孙康[1] 张积华[2] 张才龙[1] 刘世海[3] 周明[1] 吕江涛[1] 

机构地区：[1]青岛大学医学院附属医院关节外科,青岛266000 [2]青岛大学医学院附属医院查体中心,青岛266000 [3]青岛大学医学院附属医院中心实验室,青岛266000

出　　处：《中国修复重建外科杂志》2011年第10期1250-1255,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：山东省科技攻关计划资助项目(2008GG30002037)~~

摘　　要：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 study the effect of platelet lysate（PL） on chondrogenic differentiation of human umbilical cord derived mesenchymal stem cells（hUCMSCs） in vitro.Methods Umbilical cords were voluntarily donated by healthy mothers.The hUCMSCs were isolated by collagenase digestion and cultured in vitro.The surface markers of the cells were detected by flow cytometer.According to different components of inductive medium,the cultured hUCMSCs were divided into 3 groups： group A [H-DMEM medium,10% fetal bovine serum（FBS）,and 10%PL];group B [H-DMEM medium,10%FBS,10 ng/mL transforming growth factor β1（TGF-β1）,1 × 10-7 mol/L dexamethasone,50 μg/mL Vitamin C,and 1% insulin-transferrin-selenium（ITS）];and group C（H-DMEM medium,10%FBS,10 ng/mL TGF-β1,1 × 10-7mol/L dexamethasone,50 μg/ mL vitamin C,1%ITS,and 10%PL）.The hUCMSCs were induced in the mediums for 2 weeks.Toluidine blue staining was used to detect the secretion of chondrocyte matrix.Immunofluorescence method was used to identify the existence of collagen trpe II.The expressions of Aggrecan and collagen type II were detected by semiquantitative RT-PCR.Results Flow cytometer results showed that the hUCMSCs did not express the surface markers of hematopoietic cell CD34,CD45,and human leukocyte antigen DR,but expressed the surface markers of adhesion molecule and mesenchymal stem cells CD44,CD105,and CD146.Toluidine blue staining and immunofluorescence showed positive results in group C,weak positive results in group B,and negative results in group A.Semiquantitative RT-PCR showed the expressions of Aggrecan and collagen type II at mRNA level in groups B and C,but no expression in group A.The mRNA expressions of Aggrecan and collagen type II were higher in group C than in group B（P 〈0.05）.Conclusion Only 10%PL can not induce differentiation of hUCMSCs into chondrocytes,but it can be a supplement to the induced mediums.PL can improve hUCMSCs differentiating into chondrocytes obviously in vitro.This study provides new available conditio

关 键 词：组织工程软骨 血小板裂解液 人脐带间充质干细胞 软骨细胞 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]