论文部分内容阅读
目的:观察人脐带间充质干细胞体外长期培养的生物学特性,探讨其向肝样细胞分化的可能性。方法:脐带来源于天津市第三中心医院住院的健康足月产妇,采用酶消化法分离培养人脐带间充质干细胞,待细胞生长至80%～90%融合时传代。取传至第9代细胞接种于12孔板内,调整细胞浓度为5×1010L-1,加入含肝细胞生长因子、成纤维生长因子4、抑瘤素的DMEM培养基,诱导28d。MTT法测定细胞生长活性,流式细胞仪检测细胞周期,免疫细胞化学及流式细胞仪鉴定细胞表型;免疫细胞化学染色鉴定诱导后肝细胞特异表面标志物的表达,以及糖原染色结果。结果:从人脐带中分离得到的间充质干细胞贴壁生长,形态类似于成纤维细胞,80%以上处于G0/G1期,具有良好的生长活性,可在体外稳定传代20次以上;CD29,CD90,CD105,Vimentin呈阳性表达,基本不表达造血细胞标志CD34和内皮细胞标志CD31。随诱导时间的延长,圆形细胞逐渐增多,形态似肝细胞;诱导1周时细胞开始表达肝细胞表面标志物甲胎蛋白、细胞胶蛋白18、细胞胶蛋白19;诱导3周时开始表达成熟肝细胞标志白蛋白;诱导4周时白蛋白表达增强,且诱导细胞糖原染色呈阳性。结论:从人脐带中可成功分离出间充质干细胞,实现体外长期培养,并可诱导分化为肝样细胞。
OBJECTIVE: To observe the biological characteristics of human umbilical cord mesenchymal stem cells cultured in vitro for a long time and to explore the possibility of their differentiation into hepatocyte-like cells. Methods: The umbilical cord originated from healthy full-term pregnant women hospitalized in the Third Central Hospital of Tianjin. Human umbilical cord mesenchymal stem cells were isolated and cultured by enzymatic digestion. When the cells grew to 80% -90% confluence, they were passaged. The cells of passage 9 were inoculated into 12-well plates and adjusted to a cell concentration of 5 × 1010 L-1. DMEM medium containing hepatocyte growth factor, fibroblast growth factor 4 and oncostatin was added and induced for 28 days. Cell viability was measured by MTT assay. Cell cycle was detected by flow cytometry. Immunocytochemistry and flow cytometry were used to identify the cell phenotype. Immunocytochemical staining was used to identify hepatocyte specific surface markers and glycogen staining. Results: The mesenchymal stem cells isolated from human umbilical cord adherently grew in the form of fibroblasts. More than 80% of them were in G0 / G1 phase and had good growth activity, which could be stably passaged more than 20 times in vitro. CD29, CD90, CD105, Vimentin positive expression, the basic does not express the hematopoietic cell marker CD34 and endothelial cell marker CD31. With the prolongation of the induction time, the number of round cells increased gradually, and the morphology was similar to that of hepatocytes. At 1 week after induction, the cells began to express hepatocyte surface markers α-fetoprotein, Cytokeratin 18 and Cyto 19. At 3 weeks, the cells began to express mature Hepatocyte marker albumin; albumin expression enhanced 4 weeks after induction, and induced glycogen staining of cells was positive. CONCLUSION: Mesenchymal stem cells can be successfully isolated from human umbilical cord, long-term cultured in vitro and induced to differentiate into hepatocyte-like cells.