摘要
目的: 应用荧光蛋白标记细胞的技术, 对组织工程软骨体外构建过程进行活细胞的实时示踪。方法: 采用逆转录病毒载体pLEGFP N1对人间充质干细胞进行荧光蛋白标记, 以同期培养未标记细胞为对照组, 体外构建细胞 材料复合体, 通过倒置荧光显微镜和扫描电镜观测种子细胞在支架材料内的分布与数量, 并对培养 7d的两组复合体进行细胞数量比较。结果: 逆转录病毒载体pLEGFP N1成功标记间充质干细胞, 并应用于工程化组织体外构建的示踪; 第 7d标记组细胞数 (3. 4±0. 1)×106 /个复合体, 未标记组细胞数 (3. 6±0. 1)×106 /个复合体, 两组数据无统计学差异(P=0. 339)。结论: 采用逆转录病毒介导方式对种子细胞进行荧光蛋白标记, 不影响细胞在材料内的增殖, 对体外构建组织工程软骨具有良好的示踪作用。
Objective:To establish the new technique of labelled seeding cells with fluorescent protein for monitoring the process of construction tissue engineering cartilage in vitro. Method:Human mesenchymal stem cells (MSCs) were isolated and culture expanded from femur marrow.MSCs were transfected with the recombinant retrovirus pLEGFP N1 containing enhanced green fluorescent protein (EGFP) in experiment group. The unmarked MSCs were in control group. The scaffold was prepared with poly L lactic acid (PLLA), calcium polyphosphate fiber (CPPf). The cell scaffold complexes were constructed with fluorescent labelled MSCs and the absorbable scaffold in vitro. The tissue development was observed by fluorescence microscope and scanning electron microscopy. The number of seeding cells in the complex was analysed after digestion with trypsin and EDTA on 7 th day.Result: EGFP was successfuflly expressed after MSCs being transfected with the retroviral vector pLEGFP N1. The labelling cells were easy to be observed by fluorescence microscope while the cell scaffold complexes were constructed and cultured. After the 7 th day, the number of cells in each complex wasn′t significantly difference ( P <0.01)in experiment group [(3.4±0.1)×10 6] and in control group [(3.6±0.1)× 10 6]. Conclusion:After MSCs were transfected with the recombinant retrovirus containing EGFP, the distribution and proliferation of cells weren′t effect. It′s useful for monitoring the process of construction tissue engineering cartilage in vitro by the application of this technique.
出处
《中国矫形外科杂志》
CAS
CSCD
北大核心
2005年第2期137-140,共4页
Orthopedic Journal of China
基金
国家自然科学基金资助项目 (编号 30270375
30300079)
全军十五医药卫生科研重点基金资助项目 (编号01Z072)
关键词
间充质干细胞
组织工程
荧光蛋白
Mesenchymal stem cells
Tissue engineering
Fluorescent protein
