小鼠胚胎干细胞-聚酯复合物在体多分化特性的初步实验观察  

Preliminary in vivo study of pluripotent differentiation of murine embryonic stem cells in the presence of polymer

作　　者：马兴[1] 胡蕴玉[1] 颜永年[2] 熊卓[2] 王哲[1] 吕荣[1] 王军[1] 徐新智[1] 李丹[1] 

机构地区：[1]第四军医大学西京医院全军骨科研究所,西安710032 [2]清华大学机械系激光快速成形制造中心

出　　处：《中国骨质疏松杂志》2005年第4期449-452,489,共5页Chinese Journal of Osteoporosis

基　　金：国家自然科学基金资助项目(30300356);国家博士后科学基金资助项目(2003033523);西京医院科技创新基金资助项目(XJCX03M005)

摘　　要：目的初步探讨胚胎干细胞聚酯复合物在体多分化特性。方法取BALBc孕鼠3.5d的囊胚,制备内细胞团(ICM)细胞用鼠胚成纤维细胞(MEF)饲养层及条件培养液培养,挑选胚胎干细胞(ES)集落并扩增传代。应用先进快速成形技术(RP)制备聚乳酸聚羟乙酸(PLGA)人工聚酯载体,扫描电镜(SEM)观察其超微结构,并对3mm×3mm×3mm大小的粒度均匀的颗粒型聚酯载体进行Ⅰ型胶原表面修饰。取10只裸鼠,于裸鼠体部左侧皮下植入2粒Ⅰ型胶原表面修饰的颗粒型PLGA聚酯载体并同时复合ES细胞(1×107个)作为实验侧;于裸鼠体部右侧对应部位皮下仅植入2粒Ⅰ型胶原表面修饰的颗粒型PLGA聚酯载体作为对照侧,术后30d取材组织学染色对比观察。结果制备和筛选出生长旺盛、传代稳定的ES细胞。先进RP制备的PLGA聚酯载体具有规则的空间网状支架结构、相互贯通的孔隙及材料表面微孔特征。组织学发现,实验侧植入体内30d,能形成包含各胚层细胞成份的“畸胎瘤”样组织,并可观察到较为典型的软骨细胞、软骨基质以及新生骨,且在多分化新生组织形成的同时载体材料逐渐降解;对照侧术后30d未见多分化新生组织形成,并仍有相对较多的聚酯载体有待降解。实验侧的多分化新生组织形成能力、生物相容性以及聚酯载体的生物可降解性均明显优于对照侧(P<0.01)。结论先进RP技术结合载体材料表面修饰的新工艺,对ES细胞全能分化特性无明显不利影响,将有望适用于以ES细胞为种子细胞的骨、软骨组织工程构建。Objective To investigate the pluripoteut differentiation of murine embryonic stem cells（ES） in the presence of polymer. Methods The 3.5 d BALB/c murine embryos were collected and cuhured on the murine embryonic llbroblast（MEF） feeder layers. Murine ES cells were isolated from inner cell mass and cultured. Threedimensional poly DL-lactic-co-glycolic acid （PLGA） scaffolds were manufactured by rapid prototyping （RP） technique. The microstructure of PLGA scaffolds was observed by scanning electron microscopy. The porous PLGA scaffolds with the volume of 3 mm × 3 mm × 3 mm were coated with collagen type Ⅰ . Ten nude mice underwent bilaterally subcutaneous implantation. Two pieces of collagen type I coated PLGA scaffolds were simultaneously implanted with 1 × 10^7 ES cells on the left side of each animal. On the right side, only 2 pieces of collagen type Ⅰ coated PLGA scaffolds were implanted. Ten animals were killed 30 days after implantation and the pluripotent differentiation of ES cells in vivo was evaluated. Results Murine ES cells were obtained and RP-based PLGA scaffolds with ideal biomaterial properties such as satisfactory microarchitectures and porosities could be efficiently coated with collagen type Ⅰ. Histological analysis showed collagen type Ⅰ coated PLGA scaffolds were biocompatible and biodegradable. ES cells in the presence of the scaffolds retained their pluripotential of differentiation. The newly formed chondrocytes, cartilage and bone-like extracellular matix were found in the mass. In the controls, however, only polymer debris and fiber tissues were predominantly found（ P 〈 0.01 ）. Conclusions RP-based porous PLGA scaffolds efficiently coated with collagen type Ⅰ have a biocompatibility with ES cells.

关 键 词：胚胎干细胞 快速成形 小鼠 聚酯 复合物 分化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]