摘要
间充质干细胞(mesenchymal stem cell,MSC)是具有自我复制和多向分化潜能的一类多能干细胞。目前成人骨髓是MSC的主要来源。骨髓间充质干细胞的获取须行骨髓穿刺术,给供者造成一定痛苦,且骨髓间充质干细胞的增殖及分化潜能随供者年龄的增大而下降,因此,寻找新的MSC来源成为国内外干细胞研究领域的热点。本研究从足月胎儿脐带中分离出MSC,对其生物学特征、免疫学特征及抗急性移植物抗宿主病(acute graft versus host disease, aGVHD)的作用进行系统研究,旨在寻找MSC的新来源。
本实验第一部分建立从脐带中分离MSC的方法,并与成人骨髓源MSC对比,研究脐带源MSC的生物学特征。结果显示,脐带源MSC的分离成功率达100%;与骨髓源MSC相比,脐带源MSC成纤维细胞集落形成单位(CFU-F)比例、增殖能力和神经细胞诱导分化能力均高于骨髓源MSC,HLA-I和CD106分子表达低于骨髓源MSC(P<0.05)。脐带源MSC形态、大多数分子表型(CD13、CD29、CD44、CD105、CD73、CD166阳性,CD14、CD34、CD38和CD45阴性,CD31阴性)、细胞周期状态(超过80%的细胞处于G_0/G_1期)、脂肪和骨诱导分化能力、细胞因子分泌(表达SCF、TPO、FL、IL-6、M-CSF、LIF、SDF-1和VEGF,不表达IL-3)和长期支持造血的能力与骨髓源MSC相似。
本实验第二部分研究脐带源MSC的免疫学特征。免疫表型分析显示,脐带源MSC表达HLA-I,不表达HLA-DR,不表达协同刺激分子CD80、CD86和CD40。3H-胸腺嘧啶核苷(3H-Tdr)掺入法检测淋巴细胞增殖结果显示,脐带源MSC不刺激小鼠淋巴细胞增殖,且可显著抑制小鼠混合淋巴细胞反应。
本实验第三部分通过GVHD小鼠模型初步研究了脐带源MSC对aGVHD的影响。对GVHD鼠生存时间、GVHD靶器官(肝脏、小肠和皮肤)病理改变、淋巴细胞亚群和细胞因子水平的研究结果证明,脐带源MSC静脉输入可显著减轻小鼠半相合移植模型aGVHD的严重程度。其机制可能与增高Th2细胞因子
Mesenchymal stem cells (MSCs) are of great therapeutic potential due to their capacity of self-renewal and multilineage differentiation. Currently, bone marrow (BM) represents the major source of MSCs for cell therapy. However, aspiration of BM involves invasive procedure. Moreover, the frequency and multi-lineage differentiation potential of BM-MSCs decrease significantly with age. Therefore, the search for alternative sources of MSCs is of significant value. We isolated abundant MSCs from the full term umbilical cord (UC). The biological characteristics, the immunological function and the effect on graft-versus-host disease (GVHD) of the UC-MSCs were further determined. The aim of this study is to find the alternative source for BM-MSCs.
In the first part, a method to isolate abundant MSC from UC was described. The biological characteristics of the UC-MSCs were further determined and compared with BM-MSCs. The results showed that MSCs were successfully isolated from all the 36 UC and 8 BM. Although the mean number of nucleated cells isolated of UC was significantly lower than that of BM (1×10~6 /cm vs. 5.5×10~7/ml) (p=0.0002), no significant differences of the yield of adherent cells were observed (8.6×105/cm vs. 8.4×10~5/ml) (p>0.05). UC-MSCs shared the most of the characteristic of BM-MSCs, including fibroblastic-like morphology, typical immunophenotype, cell cycle status,
引文
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