自体软骨细胞复合Ⅰ型胶原支架体外动态培养的实验研究  

In vitro culture of chondrocytes in combination with typeⅠcollagen scaffold in a micro- gravity bioreactor: anexperimental study

作　　者：王泽民[1] 段莉[2] 梁宇杰[3] 王大平[2] 

机构地区：[1]广州医科大学,广州510182 [2]广东深圳市第二人民医院,518035 [3]北京大学深圳研究生院,深圳518035

出　　处：《中国矫形外科杂志》2015年第18期1693-1698,共6页Orthopedic Journal of China

基　　金：广东省自然科学基金项目(编号:S2012010008129);深圳市科技创新委技术攻关项目(编号:JSGG2014051905550503);深圳市科技创新委国际科技合作项目(编号:GJHZ20130412159306739);深圳市重点实验室提升项目(编号:CXB201104220049A);深圳市科技研发资金项目(编号:CXZZ20120614160234842;ZDSY20120614154551201);中国博士后科学基金面上项目(编号:2013M530385)

摘　　要：[目的]比较体外静态与动态培养两种方式对培养人关节软骨细胞复合Ⅰ型胶原支架材料的效果,从而探索体外构建移植物用于治疗关节软骨缺损的适宜条件及方式。[方法]分离培养人关节软骨细胞,P2代软骨细胞接种于Ⅰ型胶原支架,随机分为3组,A组:静态培养;B组:动态培养;C组:单层培养。采用倒置相差显微镜、荧光显微镜、SEM、HE染色观察细胞在支架上的分布、黏附、生长及形态特点,实时荧光定量PCR检测软骨细胞表型特异基因Ⅱ型胶原的表达情况。[结果]体外单层培养的软骨细胞(P2)以多角形为主,Ⅱ型胶原蛋白表达阳性,提示P2代细胞表型维持良好;与A组比较,B组细胞数量较多且分布均匀,细胞形态较均一,以多角形为主;A、B组样本大体结构及内部孔隙结构均保持完整,孔隙内均可见细胞黏附,B组孔隙内细胞数量及细胞外基质优于A组;A、B组的Ⅱ型胶原基因mRNA表达水平较单层培养组明显增加。[结论]软骨细胞复合Ⅰ型胶原支架体外动态培养较静态培养可明显促进细胞增殖及细胞外基质分泌。因此,动态培养有望成为体外构建自体软骨细胞移植术所需移植物的一种有效方法。The aim of this study is to compare the effects between static and dynamic system on human articu-lar chondrocytes in combination with type I collagen scaffold, thus providing a proper way to culture transplants for cartilage defect repair. [ Method ] Human chondrocytes were isolated from articular cartilage and sub - cloned at 90% confluence. Chondrocytes （P2） were seeded on type I collagen scaffolds and randomly assigned to 3 groups： group A （ static culture）, group B （dynamic culture）, and group C was set as the blank control （ mono - layer culture ）. The distribution, adhesion, proliferation, and morphology of chondrocytes on scaffolds were detected by inverted phase contrast microscope, fluores- cence microscope, SEM and HE staining. The expression level of type II collagen （ COL - 2 ） , as a specific marker of chon- drocyte phenotype, was determined by real time pelymerase chain reaction （ RT - PCR）. [ Result] The typical morphology of chondrocytes （ P2 ） was polygonal when cultured in monolayer. The COL -2 protein was positively expressed. Thus, P2 chondrocyte phenotype was well maintained. Compare to that of group A,more polygonal chondrocytes were evenly distributed on the scaffold in group B. The macro -structure and inner pore struc- ture kept intact in a 3 - Dimentional （3 - D） culture （ group A and B）. The number of chondrocytes in the pores and the quan- tity of secreted extracelluar matrix were significantly improved in group A than that in group B. In addition, compared to 2 - D culture,3 -D culture resulted in higher expression level of COL-2 mRNA. [ Conclusion] Dynamic culture in a micro- gravity system has demonstrated advantages over static culture, being able to promote chondrocyte proliferation and extracelluar matrix formation. Therefore,the dynamic culture strategy and system used in this study could be an efficient way to culture transplants for cartilage repair.

关 键 词：软骨细胞 I型胶原支架材料 动态培养 

分 类 号：R318.08[医药卫生—生物医学工程]