【摘要】 目的 :探讨采用含淤胆血清的培养体系从骨髓细胞中筛选与扩增骨髓源性肝干细胞的可行性。方法 :制备含 2 %、5 %、7%和 10 %淤胆血清的筛选培养液 ,培养大鼠骨髓细胞 ,4d时加入肝细胞生长因子促进肝干细胞生长 ,2周时行免疫组化与RT -PCR检测肝干细胞标志、糖原染色和尿素合成试验分析功能。结果 :在 2 %淤胆血清中 ,骨髓细胞不能形成克隆 ,7%与 10 %的血清则骨髓细胞逐渐凋亡。在 5 %的淤胆血清时 ,骨髓源性肝干细胞在此病理筛选培养液中能够生存并选择性生长 ,而其他类型细胞则脱落凋亡 ;第 2周时 ,形成肝细胞样集落 ,细胞表达胚胎时期肝细胞特征性蛋白 ,具有肝细胞特有糖原和尿素合成功能。结论 :含淤胆血清的微环境筛选培养体系能够有效地从骨髓细胞中筛选骨髓源性肝干细胞 ,为临床肝细胞替代治疗的获取丰富供体细胞来源提供了新的思路。更多还原

【Abstract】 AIM: To explore the feasibility of direct separat and selective enlargement of the bone marrow-derived liver stem cells (BDLSC) from bone marrow cells with a culture system containing cholestatic serum in vitro . METHODS: Bone marrow cells of rats were cultured with selective media containing 2%, 5%, 7% and 10% cholestatic rat serum, respectively. The BDLSC were then induced to proliferate with the addition of hepatocyte growth factor (HGF) on the firth day. BDLSC were characterized using immunocytochemistry and RT-PCR for lineage markers, glycogen staining and urea synthetic assay for functions 2 weeks later. RESULTS: Bone marrow cells were unble to form colony in the presence of 2% cholestatic serum and apopotosis appeared gradually in 7% or 10% cholestatic serum. The BDLSC survived in the medium containing 5% cholestatic serum while the other types of cells did not. The survival cells proliferated with a high speed during the second week and then formed hepatocyte-like colony-forming units (H-CFU). Cells in the H-CFU expressed the characteristic proteins of fetal hepatocytes. Furthermore, they had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes. CONCLUSION: The selective micro-environment effectively selected BDLSC from the bone marrow cell, and will be a new way to provide an abundant source of donor hepatocytes for clinical cell therapy.更多还原