软骨细胞膜片的成软骨性能  

Chondrogenesis of chondrocyte cell sheets

作　　者：周丽斌[1] 吴玮[1] 丁瑞英[1] 徐冰心[1] 韩浩伦[1] 王刚[1] 李保卫[1] 王鸿南[1] 

机构地区：[1]解放军第306医院耳鼻咽喉头颈外科,北京市100101

出　　处：《中国组织工程研究》2017年第12期1843-1848,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金青年科学基金项目(81401609)~~

摘　　要：背景:基于温敏培养皿的细胞膜片技术在组织工程研究中受到越来越多的关注,但是温敏培养皿价格高昂,有必要研究替代方法。目的:应用普通培养皿进行细胞膜片培养,并分析软骨细胞膜片的成软骨性能。方法:取1例鼻中隔偏曲患者手术切除的鼻中隔软骨消化提取原代软骨细胞,扩增培养。取第3代细胞在体外密集培养,使之分泌细胞外基质,形成单层细胞膜片;另取第2代软骨细胞,叠加接种于单层细胞膜片上,使其贴覆生长,形成双层细胞膜片。应用细胞刮刀收获细胞膜片,研究其性状,进一步在体外培养、植入裸鼠体内构建出人工软骨块。结果与结论:(1)单层细胞膜片和双层细胞膜片均形成质软的凝胶样结构,大体观察两者无明显区别;(2)新刮起的细胞膜片具有良好的可流动性和成胶性;植入裸鼠体内培育8周之后,均形成成熟的软骨块;(3)组织学观察见细胞膜片是由成层排列的软骨细胞和细胞外基质组成的薄片,在体外培养过程中即有糖胺聚糖和Ⅱ型胶原的初步表达,植入体内8周之后形成的软骨呈现为成熟的软骨组织;(4)生化定量检测结果表明,DNA含量高于天然鼻中隔软骨,而糖胺聚糖和羟基脯氨酸含量与天然软骨差异无显著性意义;(5)结果提示,应用普通培养皿可成功构建并获取细胞膜片,细胞膜片呈现良好的成软骨性能。BACKGROUND：The cell-sheet technology, based on a temperature-responsive culture, has been drawing more and more attention;however, the temperature-responsive culture dish is quite expensive. Therefore, it is imperative to develop a substitutive technique.OBJECTIVE： To study the feasibility of cell-sheet ulturing using common culture dish, and investigate the chondrogenesis of the cell sheet. METHODS： A piece of nasal septal cartilage was adopted from a patient with deviation of nasal septum to extract primary chondrocytes that were then cultured and amplified. The passage 3 chondrocytes were used to construct ell sheets. Monolayer cell sheet was formed by intensive culturing and allowing the extracellular matrix secretion. Bilayer cell sheet was constructed by seeding passage 2 chondrocytes on the monolayer cell sheet. The cell sheets were harvested using cell scraper, their properties were investigated prior to plantation into nude mice to construct the tissue-engineered cartilage. RESULTS AND CONCLUSION： Both bilayer and monolayer cell sheets with soft tremellose structures showed no significant difference through naked eyes. The newly harvested cell sheets appeared to have good fluidity and gelation. Eight weeks after mplantation into the nude mice, mature cartilage blocks were obtained. Histologically, the cell sheets were thin films composed by layered chondrocytes and extracellular matrix. Glycosaminoglycan formation and type Ⅱ collagen expressions were observed in the cell sheets cultured in vitro. The explanted samples exhibited ature cartilaginous tissue at 8 weeks after implantation. Biochemical analysis showed that the DNA contents of the neocartilages were higher than those of native human costal cartilage, while the contents of glycosaminoglycan and hydroxyproline were similar to native human nasal septal cartilage. To conclude, the hondrocyte cell sheets are likely to be constructed and harvested successfully using common culture dish, and the cell sheets exhibit favourable chondrogenesis.

关 键 词：组织构建 软骨组织工程 细胞膜片 软骨 培养 普通培养皿 糖胺聚糖 Ⅱ型胶原 成软骨性能 国家自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]