成肌干细胞在透明质酸凝胶载体中诱导分化为软骨细胞的研究  

Experimental study of myoblasts differentiating into chondrocytes in gel of hyaluronic acid

作　　者：李全双[1] 梅晰凡[1] 郭占鹏[1] 王滢丽[2] 秦书俭[3] 

机构地区：[1]辽宁医学院附属第一医院骨科,辽宁锦州121000 [2]辽宁医学院附属第一医院内分泌科,辽宁锦州121000 [3]辽宁医学院解剖教研室,辽宁锦州121000

出　　处：《解放军医学杂志》2010年第7期829-832,共4页Medical Journal of Chinese People's Liberation Army

基　　金：辽宁省博士启动基金(20081100)

摘　　要：目的探讨以透明质酸钠凝胶作为组织工程软骨载体、以成肌干细胞作为种子细胞诱导分化形成软骨细胞的可行性。方法体外分离培养兔成肌干细胞,采用结蛋白(Desmin)细胞化学染色进行鉴定,并观察兔成肌干细胞在不同浓度透明质酸钠凝胶中的生长状态。在5%透明质酸钠凝胶中以骨形态发生蛋白2(BMP-2)诱导兔成肌干细胞,倒置相差显微镜观察细胞生长情况,并通过甲苯胺蓝染色、免疫细胞化学及RT-PCR技术对诱导后的细胞进行鉴定。结果原代培养兔成肌干细胞Desmin细胞化学染色呈阳性表达,且在不同浓度透明质酸钠凝胶中均生长良好。BMP-2诱导培养1d后,成肌干细胞即开始于透明质酸钠凝胶中伸展生长,诱导培养10d后,细胞形态由梭形向多边多角形转化,甲苯胺蓝染色阳性,Ⅱ型胶原免疫荧光检测阳性,RT-PCR检测Ⅱ型胶原和聚焦蛋白聚糖mRNA呈阳性表达。结论兔成肌干细胞可在适当浓度BMP-2诱导下分化为软骨样细胞,透明质酸钠凝胶可作为软骨组织工程的良好载体。Objective To investigate the feasibility of using sodium hyaluronate gel as a tissue engineering scaffold and myoblasts as seed cells to induce formation of chondrocytes.Methods Rabbit myoblasts were isolated and cultured in vitro and identified by cytochemical staining of desmin.The growth status of rabbit myoblasts in different concentrations （2.5%,5% and 10%） of sodium hyaluronate gel was observed by phase microscopy and the cell viability was detected by MTT method.The rabbit myoblasts were induced with bone morphogenetic protein-2 （BMP-2） in 5% sodium hyaluronate gel,and the growth status was observed by inverted phase microscopy,and the induced cells were identified by toluidine blue staining,immunocytochemical analysis and RT-PCR technique.Results The desmin cytochemical staining of primary rabbit myoblasts was positive.The rabbit myoblasts grew well in different concentrations of sodium hyaluronate gel.One day after BMP-2 induction,growth of the myoblasts with extension was observed in sodium hyaluronate gel.Ten days after BMP-2 induction,the cells transformed from spindle to polygonal shape,the toluidine blue staining and collagen type Ⅱ staining were positive,and collagen type Ⅱ and aggrecan mRNA were detected by RT-PCR.Conclusion Rabbit myoblasts may differentiate into chondrocytes in proper concentration of BMP-2,and sodium hyaluronate gel can be used as a favorable scaffold for chondrocytes tissue engineering.

关 键 词：成肌细胞 骨形态发生蛋白质类 透明质酸 细胞分化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]