大鼠骨髓间充质干细胞的生物学特性和示踪标记

Biological properties and trace labeling of rat bone marrow mesenchymal stem cells

目的 建立大鼠骨髓间充质干细胞（MSCs）的分离和培养方法,观察绿色荧光蛋白（GFP）基因通过慢病毒载体感染MSCs的表达.方法 采用原代贴壁法获得骨髓MSCs,观察细胞形态和生长变化,流式细胞仪鉴定细胞表面标志,体外诱导MSCs向脂肪细胞和成骨细胞分化.采用含有增强型绿色荧光蛋白（EGFP）基因慢病毒载体感染培养的MSCs,比较细胞感染前后生物学特性.结果 原代贴壁筛选结合差异传代培养的MSCs表面标志阳性率分别为CD44 94.81%,CD90 99.53%,CD106 76.34%,MSCs在pH值稳定于7.2～7.4的环境可传20代.体外MSCs诱导分化后特异性染色显示脂质沉淀和骨结节,表达脂肪细胞和成骨细胞特异基因.慢病毒载体可有效感染大鼠MSCs,加入聚凝胺感染效率达到80%,EGFP在MSCs感染后1个月仍持续表达.结论 骨髓MSCs可长期培养,具有良好的多向分化能力.携带EGFP基因慢病毒载体能高效感染MSCs,EGFP可作为MSCs体内研究的示踪标记.

Objective To establish a method of isolation and culture of the rat mesenchymal stem cells （MSCs） , and to investigate the expression of green fluorescent protein （GFP） gene carried by lentiviral vectors into MSCs. Methods MSCs, which were initially isolated from the bone marrow of rats, were cultured in vitro, isolated by trypsin digestion method, purified by adherence method and tested by flow cytomertry. After MSCs were transferred to osteogenic and adipogenic medium respectively, the morphological characterization of induced cells was observed. The expression of marker genes was measured by reverse transcription-polymerase chain reaction （RT-PCR）. MSCs were infected with lentiviral vectors. The results of the expression of GFP and infection efficiency were observed by fluorescence microscope. Results MSCs of high purity were obtained from bone marrow using adherence screening method. The stable pH of culture medium between 7.2-7.4 helped MSCs having subculture of 20 generations. MSCs typically expressed the antigens CD44 （94.81%） , CD90 （99.53% ） and CD106 （76.34%）. They were negative for typical lymphocytic markers like CD45 （ 1.94% ） and CD11b （ 1.42% ） and for the early hematopoietic markers CD34 （0.04%）. The infection rate of lentiviral vector in medium containing polybrene could reach 80%. The exogenous EGFP and multilineage potential of MSCs had no severe influences on each other. Conclusion Since the MSCs can be easily obtained and abundant, it is proposed that they may be promising candidate cells for further studies on tissue engineering. Imaging with expression of EGFP facilitates the research on MSCs physiological behavior and application in tissue engineering during differentiation both in vitro and in vivo.