骨髓源性肝干细胞的筛选、扩增和分化

目的 探讨采用含淤胆血清的培养体系直接从体外全骨髓细胞培养中筛选、扩增和分化骨髓源性肝干细胞的可行性.方法 制备含不同浓度淤胆血清的条件筛选培养液,常规培养大鼠全骨髓细胞,贴壁后换用条件筛选培养液,根据筛选的结果确定最佳的淤胆血清浓度.筛选到的骨髓源性肝干细胞分别采用扩增培养液和分化培养液进行扩增和诱导分化.传代细胞应用流式细胞仪检测干细胞标记.采用免疫组织化学、RT-PCR和电镜等方法对骨髓源性肝干细胞进行形态学以及表型特征的鉴定.以糖原染色和尿素分析的方法对诱导分化的细胞进行代谢功能的测定.结果 筛选培养结果,含50 ml/L的淤胆血清培养液的筛选效果最佳:骨髓源性肝干细胞能够生存,而其他非肝干细胞因不能适应而凋亡.纯化的肝干细胞能在扩增培养体系中传代6代并且维持稳定的细胞表面特征.更换为分化培养体系后,可形成肝细胞样集落形成单位.肝细胞样集落形成单位的细胞表达胎肝细胞的标志(AFP,白蛋白和细胞角蛋白8/18),胆管细胞的标志(细胞角蛋白19),肝细胞的功能蛋白(甲状腺素转运蛋白和细胞色素P450-2 b1),以及肝细胞核因子(HNF-1α和HNF-3 β).同时具有糖原储存和尿素合成等肝细胞特有功能.结论 含淤胆血清的筛选培养液能从全骨髓细胞培养中有效地筛选骨髓源性肝干细胞,并且纯化的肝干细胞能传代6代.肝干细胞分化后能形成具有肝细胞样的表型和功能.骨髓源性肝干细胞为解决临床肝细胞治疗的肝细胞来源问题提供了一个新的方法.

Selection, proliferation and differentiation of bone marrow derived liver stem cells

Objective To explore the feasibility of direct separation, selective proliferation and differentiation of BDLSC from hone marrow cells with culture systems containing cholestatic serum in vitro.Methods Whole bone marrow cells of rats cultured in routine medium were replaced with con-ditioning selection media containing eholestatic sera of different concentrations after they attached to the plates.The optimal concentration of cholestatic serum was determined according to the outcome of the selected cultures.Then the selected BDLSC were induced to proliferating culture system and dif-ferentiating culture system.Each passage of the proliferated stem cells was subjected to flow cytome-try for detection of stem cell markers.The morphology and phenotypie markers of BDLSC were char-acterized using immunohistochemistry, RT-PCR and electron microscopy.The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay.Results The 50 ml/L eholestatie serum was the optimal concentration for the selection of BDLSC at which BDLSC could sur-vive while the other populations of the bone marrow cells could not.The purified BDLSC could prolif-erate for 6 passages and maintained stable markers in our proliferating system.When the culture sys-tem changed to differentiating system, hepatocyte-like colony-forming units (HCFU) were formed.HCFU expressed markers of embryonic hepatoeytes (AFP, albumin and cytokeratin 8/18), biliary cells (cytokeratin 19), hepatocyte functional proteins (transthyretin and eytoehrome P450-2b1), and hepatocyte nuclear factors (HNF-1α and HNF-3β).They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes.Conclusion BDLSC can be selected directly from whole bone marrow cells and pure BDLSC can also proliferate for 6 passages.The differentiated cells have hepatocyte-like phenotype and function.BDLSC will he a new way to provide a readily avail-able alternate source of cells for clinical hepatocyte therapy.