人脐带间充质干细胞体外成骨及其免疫学特征

In vitro osteogenesis and immunological characteristics of human umbilical cord-derived mesenchymal stem cells

背景:目前关于脐带源性干细胞的研究主要是针对其多向分化、组织修复方面,而对脐带干细胞的同种异体之间移植免疫学的报道较少。目的:观察体外培养的人脐带间充质干细胞生物学特性、成骨分化潜能及移植免疫学特征。设计、时间及地点:细胞学体外实验,于2008-11在吉林大学药学院病理学实验室完成。材料:脐带来源于健康产妇足月剖腹产的胎儿,靠近胎儿侧5cm,均征得孕妇同意。方法:应用组织块贴壁法分离培养脐带间充质干细胞,胰酶消化传代。取传至第5代细胞,加入含地塞米松、抗坏血酸、β-甘油磷酸钠的α-MEM完全培养基进行成骨诱导。另取传至第5代细胞,分别进行正常培养及应用γ-干扰素刺激48h。主要观察指标:脐带间充质干细胞形态观察、生长曲线分析、免疫表型分析。碱性磷酸酶染色观察细胞成骨分化情况。流式细胞仪检测γ-干扰素刺激后细胞免疫表型变化。结果:培养7d倒置显微镜下可见组织块边缘出现呈长梭形、多角形或三角形的贴壁细胞,且随时间延长组织块周围细胞数量逐渐增多。第2,5,9代细胞之间的生长曲线无明显变化,培养20代内未见细胞衰老及增殖速度减慢等现象发生。第2代贴壁细胞CD44,CD105均呈阳性表达,CD34,CD45均呈阴性表达。成骨诱导14d后,碱性磷酸酶染色可见胞核染成均一的淡蓝色。第5代脐带间充质干细胞HLA-ABC呈弱阳性(40.50%),HLA-DR,CD80,CD86呈阴性表达;γ-干扰素刺激48h后,HLA-ABC阳性率增加至87.44%,HLA-DR,CD80,CD86仍呈阴性表达。结论:人脐带间充质干细胞体外诱导成骨能力确切,具有类似骨髓间充质干细胞的特异性表面标记,且具有同种异体移植的低免疫原性。

BACKGROUND： Researches of umbilical cord-derived stem cells mainly study the multi-directional differentiation and tissue repair. There are a few studies concerning transplantation immunology among umbilical OBJECTIVE： To observe biological characteristics, potential of differentiating into osteoblasts and transplantation immunology characteristics of human umbilical cord-derived mensenchymal stem cells （UC-MSCs） in vitro. DESIGN, TIME AND SETTING： The cytology in vitro experiment was performed at the Laboratory of Pathology, College of Pharmacy, Jilin University in November of 2008. MATERIALS： Umbilical cores were obtained from fetus of healthy full-term parturients delivered by caesarean, 5 cm near to the fetus, following the parturients signed the informed consent. METHODS： UC-MSCs were isolated and cultured by the tissue block culture, and digested in trypsin and passaged. At passage 5, UC-MSCs were incubated in a-MEM complete medium containing dexamethasone, ascorbic acid and β-sodium glycerophosphate. Cells at passage 5 were normally incubated, and stimulated in interferon-γ for 48 hours. MAIN OUTCOME MEASURES： Morphologic characteristics, growth curve and immunophenotype of UC-MSCs were observed. Differentiation of cells into osteoblasts was observed using alkaline phosphatase staining. Immunophenotype changes of interferon-y were determined by flow cytometry following stimulation. RESULTS： Following 7 days of culture, tissues were spindle, polygonal or triangular adherent cells under an inverted microscope With the prolongation of time, the number of cells surrounding the tissue blocks was gradually increased. At passages 2, 5 and 9, no significant changes were detected in growth curve. No cell aging or decreased proliferation speed were detected within 20 passages. At passage 2, adherent cells were positive for CD44 and CD105, but negative for CD34 and CD45. Following 14 days of induction, alkaline phosphatase staining demonstrated a light blue reaction. At passage 5, UC-MSCs presented a weak positive expression of HLA-ABC （40.50%）, negative expression of HLA-DR, CD80, CD86. Following 48 hours of interferon-y stimulation, HLA-ABC positive rate increased to 87.44%, but HLA-DR, CD80, CD86 still presented negative expression. CONCLUSION： It is confirmed the potential of the differentiation of human UC-MSCs into osteoblasts in vitro. They had the similar specific surface marker as bone marrow mesenchymal stem cells, with the low immunogenicity of allogeneil graft.