摘要
目的建立人胎儿肝脏非实质细胞间充质干细胞(nonparenchymalmesenchymalstemcells,NPMSC)的分离、培养方法,观察NPMSC形态学、细胞生物学特性及细胞表型特征,以获得大量人源性间充质干细胞(MSC)。方法采用体外细胞培养技术分离培养人胎儿NPMSC,用显微摄像、MTT和图像分析研究其增殖及生长特征,用流式细胞仪和免疫组化法鉴定其表型。结果每个胎儿肝脏可获得10000±2000个贴壁细胞,可见5～10个高速增生的细胞集落。原代和传代培养均生长缓慢,按1∶3传代,传1代需8～10d,细胞经传代后逐渐纯化,传10代后可达109个细胞。流式细胞仪检测NPMSC不表达CD34,而表达CD166。对传代NPMSC进行人甲胎蛋白和白蛋白免疫组化分析,细胞表达微量甲胎蛋白,不表达白蛋白。结论肝非实质细胞中含有MSC,且量较大,可成为种子细胞。NPMSC在普通培养条件下不向肝细胞分化。
Objective To establish methods of isolation and culture for fetal liver nonparenchymal mesenchymal stem cells （NPMSCs）, and observe morphological, cytobiology and phenotypes characteristics of NPMSCs in order to provide MSCs resource. Methods Fetal liver NPMSCs were isolated and cultured with cell culture technique in vitro; the characteristics of proliferation and growth of fetal liver NPMSCs were investigated with MTT and image analysis. The phenotypes characteristics of NPMSCs were identified by flow cytometry and immunohistochemistry. Results Ten thousands ±2 000 adherent ceils were obtained from each fetus liver, there were 5 - 10 rapidly proliferative colonies. Cells of origin culture and serial subcultivafion grew slowly. Serial subcultivation was conducted on the basis of 1 to 3 ratio, the average period of serial subcultivation was 8 - 10 d, ceils were purified after serial subcultivation, the amount of ceils could reach to 109 ceils after 10 serial subcultivations. The phenotype characteristics of NPMSCs was CD166 positive and CD34 negative. Serial subculfivation NPMSCs expressed minute amount of AFP and did not express ALB. Conclusion There are certain amount of MSCs in nonparenchymal cell of liver and may be as seeds cells. Serial subcultivation NPMSCs did not differentiate to hepatocyte-like cell under common culture condition.
出处
《肝脏》
2005年第3期182-185,共4页
Chinese Hepatology
基金
国家高技术研究发展计划(863)资助项目(2001AA216161)
