单例先天性小耳残耳软骨细胞体外构建人耳廓软骨  

Generation of full-sized and ear-shaped cartilage with passaged remnant ear chondrocytes from microtia individual

作　　者：康宁 刘霞 曹谊林 肖苒 

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

出　　处：《基础医学与临床》2014年第5期583-588,共6页Basic and Clinical Medicine

基　　金：国家自然科学基金(31300801);北京市科技计划项目(D090800046609003)

摘　　要：目的 探讨单例先天性小耳残耳软骨细胞体外构建正常人大小耳廓形态组织工程软骨的可行性.方法 分离40例小耳畸形患者残耳软骨细胞统计细胞提取率;MTT法检测细胞增殖能力、计算扩增效率;免疫荧光和PCR检测不同代数细胞的软骨表型.分别应用3例患者各自的残耳软骨细胞培养至P3～P4代,藻酸盐凝胶包埋接种于正常人大小耳廓形态的聚羟基乙酸/聚乳酸支架,体外成软骨诱导动态培养10周后行组织学染色观察.结果 耳软骨细胞提取率为（3.90±1.27）×106/g;在增殖培养基中细胞增殖能力明显提高,至P4代扩增（328.4 ±50.4）倍（P＜0.05）;P3代细胞Ⅱ型胶原、蛋白聚糖表达显著减弱,至P4代消失,Ⅰ型胶原表达增强;体外培养10周,实验组形成了耳廓形态的类软骨组织,可见典型软骨陷窝,番红O、甲苯胺蓝及Ⅱ型胶原染色阳性;对照组明显变形,未形成软骨结构.结论 单例先天性小耳残耳软骨细胞经体外扩增和动态诱导培养可在体外构建正常人大小耳廓软骨.Objective To test the feasibility of in vitro generation of a full-sized and ear-shaped cartilage with remnant ear chondrocytes derived from individual microtia patient.Methods The initial cell yield of remnant ear chondrocytes was analyzed from 40 cases of microtia.Proliferation was tested by MTS and expansion efficiency was calculated.The chondrocytic phenotype altering after continuous passages was characterized by immunofluorence staining and PCR.P3 ～ P4 chondrocytes were seeded onto the PGA/PLA scaffold with the shape and full size of adult human ear.The complex was cultured with a redifferentiation system composed of chondrogenic factors,alginate gel,and a rotating culture device.Results The initial cell yield from remnant ear tissue was （3.90 ±1.27） × 106/g.The proliferative ability of remnant ear chondrocytes from P1 ～ P4 passages was enhanced by adding bFGF,and the amplification of the cells expanded to P4 could reach around （328.4 ± 50.4）folds.However,the COL Ⅱ and ACAN expressions gradually declined with passages and became negative in P4 chondrocytes whereas COLI expression showed stronger.The neo-cartilage in the experimental group maintained the ear shape well and formed cartilaginous structure with positive staining of Safranin O,Toludine Blue,and COL Ⅱ,while the control group failed to form cartilage tissue and only showed fibrous structure.Conclusions A full-sized and ear-shaped cartilage can be engineered in vitro by the passaged remnant ear chondrocytes derived from individual microtia under a redifferentiation culture system.

关 键 词：先天性小耳畸形 残耳软骨细胞 耳廓形态软骨 组织工程 

分 类 号：R318[医药卫生—生物医学工程]