人胎儿骨髓间充质干细胞的分离、培养及生物学特性鉴定

Isolation, cultivation and biological identification of human fetal marrow mesenchymal stem cells

目的建立人胎儿骨髓间充质干细胞(MMSCs)的分离、培养方法,观察MMSCs形态学、细胞生物学及细胞表型,以获得大量人源性MMSCs.方法收集12个人胎儿的骨髓,采用细胞培养技术,分离培养人胎儿MMSCs,用显微摄像、四甲基偶氮唑盐(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细胞化学鉴定其表型.结果0.5～2 h内尽快分离骨髓细胞,24 h换液后可获得约(300±80)个贴壁细胞,5个细胞以上的集落为(15±6)个;培养细胞在种植后1～3 d为生长滞留期,第4天达到对数生长期,以后进入平台期,细胞分裂指数曲线的趋势与生长曲线基本类似,在达到对数生长期后,分裂相细胞明显减少.原代及传代培养显示,细胞分裂旺盛,可观察到干细胞特有的不均等分裂,5～7 d可传1代,连续传10代后,每个人胎儿来源MMSCs可扩增达1011～1012个细胞.MMSCs表型为CD166阳性,CD34阴性.对传代MMSCs进行人甲胎蛋白、白蛋白和细胞角蛋白18免疫细胞化学分析,细胞除表达微量甲胎蛋白外,不表达白蛋白和细胞角蛋白18.结论人胎儿MMSCs分离成分单纯,容易成功,增殖旺盛,在普通培养条件下不向肝细胞分化.MMSCs作为组织工程重建的种子细胞具有较强的可行性.

Objective Noting the morphological and cytobiology characteristics and phenotypes of MMSCs, to establish an isolation and culture method for fetal MMSCs in order to provide a source of marrow mesenchymal stem cells (MMSCs). Methods Fetal MMSCs were isolated and cultured with in vitro cell culture technique; the characteristics of the proliferating and growing fetal MMSCs were studied with MTT and image analysis; the phenotypes of MMSCs were identified by flow cytometry and immunohistochemistry. Results Bone marrow of 12 fetuses was isolated within 0.5-2 h, and about 300 ± 80 adherent cells were obtained at 24 h. Colonies with more than 5 cells were 15 ± 6, growth detention period of culture cell was at 1-3 d after planting, log phase growth period was at day 4, and the amount of disintegration phase cells was reduced significantly. Original culture and serial subcultivations showed that cells divided prosperiously; unequal divisions special for stem cells were observed, and the amount of MMSCs harvested from each fetus was as much as 1011-1012 cells after 10 serial subcultivations. The phenotype of MMSCs was CD166 positive and CD34 negative. Serial subcultivated MMSCs expressed a microamount of AFP and did not express albumine or CK18. Conclusion Fetal MMSCs are easily isolated and proliferate prosperouly. Serial subcuhivated MMSCs did not differentiate into hepatocyte-like cells under common culture condition and are feasibile as seed cells for tissue engineering reconstruction.