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背景:肝细胞自身增殖能力有限,近几年关于各类干细胞向肝细胞分化的成功报道很多,包括胚胎干细胞、骨髓细胞、胰腺干细胞、神经干细胞及各种来源的间充质干细胞等。目的:探讨应用多种生长分化因子体外联合诱导人脐血间充质干细胞向肝样细胞分化的可行性。设计、时间及地点:细胞学体外观察,于2005-10/2006-04在暨南大学医学院血液病研究所完成。材料:胎儿脐带血来源于顺产及剖腹产的健康孕妇,由暨南大学第一附属医院产科提供,产妇均签署知情同意书。方法:体外分离培养人脐血间充质干细胞,胰酶-EDTA消化传代。取传至第3代细胞,按5×104/cm2接种,48h后去除原培养基,PBS洗涤后,第1阶段用含地塞米松、肝细胞生长因子、表皮生长因子、ITS的F12培养基诱导2周,第2阶段用含地塞米松、肝细胞生长因子、致瘤素M、ITS的F12培养基继续诱导2周。主要观察指标:流式细胞仪检测脐血间充质干细胞表面标志的表达,RT-PCR检测肝细胞相关基因的表达,免疫荧光染色检测肝细胞相关蛋白的表达,透射电镜观察细胞超微结构。结果:培养的细胞不表达造血细胞系标志CD34,CD45,CD14;亦不表达CD54,CD49f,HLA-DR;部分表达内皮细胞标志CD106;强表达CD29,CD44及CD13。诱导4周后,甲胎蛋白、白蛋白、ck-18及tat基因均呈阳性表达;免疫荧光染色显示细胞浆中甲胎蛋白、白蛋白、ck-18均呈阳性;细胞胞浆中出现脂滴及糖原的沉积,细胞表面有微绒毛,出现双核细胞,初步具备了肝细胞的超微结构特征。结论:联合应用地塞米松、肝细胞生长因子、表皮生长因子、致瘤素M及ITS等多种生长分化因子,可在体外成功诱导人脐血间充质干细胞向肝样细胞分化。
Background: Hepatic cells have limited ability to proliferate. In recent years, many reports have been reported on the successful differentiation of various types of stem cells into hepatocytes, including embryonic stem cells, bone marrow cells, pancreatic stem cells, neural stem cells and various kinds of mesenchymal stem cells. OBJECTIVE: To explore the feasibility of inducing human umbilical cord blood mesenchymal stem cells to differentiate into hepatocyte-like cells by using a variety of growth and differentiation factors in vitro. DESIGN, TIME AND SETTING: The cytology in vitro observation was performed at the Institute of Hematology, Jinan University Medical College from October 2005 to April 2006. MATERIALS: Fetal umbilical cord blood was obtained from pregnant women undergoing both cesarean and caesarean section. Obstetrics and gynecology was provided by the obstetrics department of the First Affiliated Hospital of Jinan University. Pregnant women both signed informed consent forms. Methods: Human umbilical cord blood mesenchymal stem cells were isolated and cultured in vitro and then passaged by trypsin-EDTA. The cells were passaged to passage 3 cells and seeded at 5 × 10 4 / cm 2. After 48 h, the primary culture medium was removed. After washing with PBS, the first stage was treated with F12 medium containing dexamethasone, hepatocyte growth factor, epidermal growth factor, ITS Induction for 2 weeks, the second phase with dexamethasone, hepatocyte growth factor, oncostatin M, ITS F12 medium continued induction for 2 weeks. MAIN OUTCOME MEASURES: Flow cytometry was used to detect the expression of surface markers of human umbilical cord blood mesenchymal stem cells. The expression of hepatocyte-related genes was detected by RT-PCR. The expression of hepatocyte-related proteins was detected by immunofluorescence staining. The ultrastructure was observed by transmission electron microscopy. Results: The cultured cells did not express hematopoietic lineage markers CD34, CD45, CD14; nor did they express CD54, CD49f, HLA-DR; partly expressed endothelial marker CD106; and strongly expressed CD29, CD44 and CD13. Four weeks after induction, the expression of alpha-fetoprotein, albumin, ck-18 and tat genes were all positive. Immunofluorescence staining showed that both alpha-fetoprotein, albumin and ck-18 were positive in the cytoplasm. Drip and glycogen deposition, cell surface microvilli, appear binuclear cells, preliminarily have the ultrastructure of liver cells. Conclusion: The combination of dexamethasone, hepatocyte growth factor, epidermal growth factor, oncostatin M and ITS and other growth and differentiation factors can successfully induce human umbilical cord blood mesenchymal stem cells to differentiate into hepatocyte-like cells in vitro.