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背景:目前研究认为在胚胎发育后的多种组织中存在一类干细胞群体,可分化为不同胚层的组织细胞;但又不同于胚胎干细胞,随着妊娠期间胚胎的发育胚胎干细胞会逐渐失去部分分化潜能,会出现一些特殊表型或分子标记,如 CD105,称其为胚胎后亚全能干细胞。目的:根据胚胎后亚全能干细胞表达 CD105的特性分离胎儿骨髓源性胚胎后亚全能干细胞,经体内外诱导使其分化为肝细胞样细胞并检测其功能。设计、时间及地点:动物随机分组,细胞分子生物学实验,于 2003-03/2005-03 在天津市第三中心医院卫生部人工细胞工程技术研究中心完成。材料:取 22 周龄左右胎儿股骨和胫骨骨髓分离出胚胎后亚全能干细胞。以雌性成年SCID 鼠作为干细胞移植的受体。CD105 免疫磁珠为德国 Miltenyi Biotec 产品。鼠抗人白蛋白抗体为美国 Sigma 产品。碱性成纤维细胞生长因子、肝细胞生长因子为英国PEPROTECH 产品。方法:利用密度梯度离心结合免疫磁珠方法分离胎儿骨髓 CD105(+)胚胎后亚全能干细胞,体外培养,用含 30 μg/L 肝细胞生长因 20 μg/L 碱性成纤维细胞生长因子的诱导培养基将其向肝细胞样细胞诱导分化。将24 只 SCID 鼠随机分为干细胞治疗组和对照组,每组 12 只,均按 800 mg/kg 的剂量向腹腔内注射 D-氨基半乳糖制备肝损伤模型。造模次日,干细胞移植组鼠在肝原位输注106 左右 CD105(+)细胞,对照组分别输注106左右的CD105(-)细胞或同等体积的培养液。主要观察指标:于干细胞移植后 2,7 d,1,3 个月对肝脏组织行免疫组化检测人源白蛋白表达。结果:免疫磁珠筛选后的细胞免疫细胞化学检测 CD105 呈弱阳性表达;细胞在对数生长期的倍增时间为 30 h 左右;约传 10 代后进入衰退期。SCID 鼠移植胚胎后亚全能干细胞 3 个月后在小鼠肝脏中可见有点状或小灶状的人白蛋白表达,对照组未见表达。结论:来源于胎儿骨髓的胚胎后亚全能干细胞可以在肝脏微环境下转化为肝细胞样细胞。
BACKGROUND: The present study suggests that there exist a group of stem cell populations in many tissues after embryo development, which can differentiate into tissue cells of different germ layers. However, unlike embryonic stem cells, embryonic stem cells gradually lose partial differentiation as embryos develop during pregnancy Potential, there will be some special phenotype or molecular markers, such as CD105, called embryonic sub-totipotent stem cells. OBJECTIVE: To isolate human mesenchymal stem cells derived from fetal bone marrow after embryonic mesenchymal stem cells express CD105, and to differentiate into hepatocyte-like cells by in vitro and in vivo induction. DESIGN, TIME AND SETTING: The animals were randomly divided into groups. Experiments in cell and molecular biology were performed at the Center of Artificial Cell Engineering and Technology, Ministry of Health, Tianjin Third Central Hospital from March 2003 to March 2005. MATERIALS: Sub-total mesenchymal stem cells were isolated from fetal femur and tibia bone marrow at 22 weeks of age. Female adult SCID mice were used as stem cell transplant recipients. CD105 immunomagnetic beads Miltenyi Biotec products. Mouse anti-human albumin antibody Sigma products in the United States. Basic fibroblast growth factor, hepatocyte growth factor for the British PEPROTECH products. METHODS: Subcultured mesenchymal stem cells of fetal bone marrow CD105 (+) embryos were isolated by density gradient centrifugation and immunomagnetic beads. The cells were cultured in vitro and induced with 20 μg / L basic fibroblast growth factor (FGF) induced by 30 μg / L hepatocyte growth. The medium induces differentiation into hepatocyte-like cells. Twenty-four SCID mice were randomly divided into stem cell treatment group and control group, with 12 mice in each group. All mice were injected intraperitoneally with D-galactosamine at a dose of 800 mg / kg to prepare a liver injury model. On the next day of modeling, rats were transfused with about 106 CD105 (+) cells in the stem of the stem cells transplantation group, and the control group were infused with about 106 CD105 (-) cells or the same volume of culture medium. MAIN OUTCOME MEASURES: Human liver albumin was detected by immunohistochemistry on day 2, day 7 and day 1, 3 after stem cell transplantation. Results: CD105 expression was weakly positive by immunocytochemistry after immunomagnetic bead screening. The doubling time of cells in logarithmic growth phase was about 30 h. After about 10 passages, the cells entered the decay phase. Three months after transplantation of SCID mouse embryonic subtypes, human stem cells showed a little or small focal area of human albumin expression in the mouse liver, but no expression in the control group. CONCLUSION: Hypodermal stem cells derived from fetal bone marrow can be transformed into hepatocyte-like cells in the liver microenvironment.