胰岛素铁硒传递蛋白促进组织工程软骨形成的作用  

Effects of insulin-transferrin-selenium on cartilage formation of chondrocytes pellets cultured in vitro

作　　者：刘瑾春[1,2] 康宁 肖苒 刘霞 曹谊林 

机构地区：[1]中国医学科学院整形外科医院研究中心,北京100144 [2]新乡医学院河南省医用组织再生重点实验室,河南新乡453003

出　　处：《新乡医学院学报》2012年第9期649-652,共4页Journal of Xinxiang Medical University

基　　金：国家自然科学基金青年科学基金项目(编号:30801192);北京市自然基金(编号:7102134);高等学校博士学科点专项科研基金(编号:200800231078);北京市科委重大计划项目(编号:D090800046609003)

摘　　要：目的研究胰岛素铁硒传递蛋白(ITS)对组织工程软骨形成的影响。方法第2代猪关节软骨细胞单层培养或离心形成细胞聚集体,根据培养液不同分为2组:常规培养组(达尔伯克改良伊格尔培养基+体积分数10%胎牛血清)和ITS组(达尔伯克改良伊格尔培养基+体积分数10%胎牛血清+体积分数1%ITS),噻唑蓝(MTT)法评估2组软骨细胞增殖;细胞聚集体培养1、2、3、4周后取材,比较2组细胞聚集体大体形态、湿质量、组织学、Ⅱ型胶原(ColⅡ)免疫组织化学染色和软骨特异基因表达。结果 MTT检测显示第5、6、7天ITS组光密度值显著高于常规培养组(P<0.01);ITS组软骨细胞聚集体体积在培养3周和4周时明显大于常规培养组;ITS组湿质量培养4周时为(7.91±1.49)mg,明显高于常规培养组的(5.37±1.31)mg(P<0.05)。ITS组细胞聚集体甲苯胺蓝染色和ColⅡ免疫组织化学染色明显强于常规培养组;2组软骨特异基因SRY相关高迁移组盒子基因9、ColⅡ表达相当,ITS组Ⅹ型胶原表达减弱,核心蛋白和Ⅰ型胶原表达增强。结论 ITS通过促进软骨细胞增殖和细胞外基质分泌可更好地促进组织工程软骨形成。Objective To investigate the effects of insulin-transferrin-selenium（ITS）on cartilage formation of chondrocytes pellets cultured in vitro.Methods Cell aggregates were formed with the second generation of pig articular chondrocytes in monolayer culture or centrifugalization.According to different culture fluid,the chondrocytes were divided two groups.Chondrocytes of conventional culture group were cultured in Dulbecco′s modified Eagle′s medium（DMEM） with 10% fetal bovine serum（FBS）;while the chondrocytes of ITS group were cultured in DMEM with 10% FBS and 1% ITS.Proliferation of chondrocytes in monolayer culture was evaluated by 3-（4,5-dimethylthiazo1-2yl）-2,5-diphenyltetrazolium bromide（MTT） assay.Chondrocytes pellets were cultured in the two groups for 4 weeks.The chondrocytes pellets were harvested at week 1,2,3 and 4,and were evaluated with histological,immunohistochemical analysis,and gene expression analysis.Results MTT assay showed that OD value in ITS group was significantly higher than that in conventional culture group（P〈0.01）.Cultured for 3 and 4 weeks,the volume of chondrocytes pellets in ITS group was significantly greater than that in conventional culture group.Cultured for 4 weeks,wet weight of pellets in ITS group was significantly higher than that in conventional culture group[（7.91±1.49） mg versus（5.37±1.31） mg,P〈0.05）].Toluidine blue staining and immunohistochemical staining of type Ⅱ collagen（Col Ⅱ） in pellets of ITS group were stronger than those in conventional culture group.There was no significant difference in the expression of SRY-related high mobility group-box gene 9 and ColⅡ gene between two groups.In ITS group,the expression of type Ⅹ collagen was decreased,but the expressions of Aggrecan and type Ⅰ collagen were increased.Conclusion ITS can promote tissue-engineered cartilage formation by promoting the proliferation and extracellular matrix secretion of chondrocytes.

关 键 词：胰岛素铁硒传递蛋白 软骨组织工程 软骨细胞 细胞外基质 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]