动态监测组织工程软骨的体外构建及三维静态培养  

Monitoring the process of construction and culture of the three-dimensional tissue-engineering cartilage in vitro

作　　者：段小军[1] 杨柳[1] 周跃 李忠[1] 陈光兴[1] 王东武[1] 李起鸿[1] 

机构地区：[1]第三军医大学西南医院关节外科中心,重庆400038 [2]新桥医院骨科

出　　处：《中国生物学文摘》2005年第10期26-29,共4页Chinese Biological Abstracts

基　　金：国家自然科学基金资助项目（30270375,30300079）,全军十五医药卫生科研重点基金资助项目（01Z072）

摘　　要：目的采用荧光蛋白标记技术，对组织工程软骨的体外构建及三维静态培养进行连续观测，从而确定适宜的体外培养时间。方法将人间充质干细胞进行荧光蛋白标记，以2×10^7个／ml的细胞密度与可吸收性软骨支架材料存体外构建细胞-材料复合体，再进行三维静态培养，通过倒置荧光显微镜对于接种后4d、1、2、3和4周的培养物，分别进行荧光强度观察和细胞数量分析。结果逆转录病毒载体pL EGFP-N1成功标记种子细胞；采用沉淀法构建细胞-材料复合体时，容易出现细胞在材料内分布不均匀的缺陷；连续培养复合体时其整体荧光强度会逐渐降低，单个复合体内的细胞数最在4d时为（1．90±0．12）×10^6，到第4周时降至（0．70±0．06）×10^6，差异有统计学意义（P＜0．01）。结沦体外静态培养组织工程软骨在4～7d时其中的细胞数量最高，适时翻转培养物有助于稳定细胞数量。Objective Application of labeled cells with fluorescent protein to monitor the process of construction and culture of the three-dimensional tissue-engineering cartilage in vitro. Methods Human mesenchymal stem cells （MSCs） isolated from femur marrow were cultuee-expanded and transfected with the recombinant retrovirus pL EGFP-N1 containing enhanced green fluorescent protein （EGFP）. The absorbable scaffold was prepared with poly-L-lactic acid （PLLA）, calcium polyphosphate fiber （CPPf）. The cell-scaffold complexes were constructed with fluorescent labeled MSCs （2×10^7/ml） and the absorbable scaffold in vitro and cultured in static condition. The tissue develcpment was observed under fluorescence microscepy. The number of seeded cells in the complex was analyzed after digestion for 4,7,14,21 or 28 days following construction, Results EGFP was successfully expressed after MSCs being transfected with the retroviral vector pL EGFP-N1. The labeled cells were easily observed under fluorescence microscopy, while the cell-scaffold complexes werc constructed and eulturcd. The distribution of cells throughout the porous scaffold wasn＇t homogeneous. The levels of expression fluorescent protein weredecreased step by step. The number of cells in each complex was （1.90±0.12）×10^6 after 4 days and （0.70±0.06）×10^6 after 28 days with the difference being significance （P 〈 0.01）. Conclusion It was useful for monitoring the process of construction tissue-engineering cartilage in vitro by the, application of labeled MSCs with fluorescent protein. The optimal time of culture of the cell-scaffold complex was from 4 days to 7 days.

关 键 词：间充质干细胞 组织工程 细胞培养 荧光蛋白 

分 类 号：Q813[生物学—生物工程] X592[环境科学与工程—环境工程]