人脐带来源间充质干细胞生物学特性的研究及其对移植物抗宿主病作用的初步探讨

发布时间：2018-08-16 10:50

【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(P0.05)。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
[Abstract]:Mesenchymal stem cells (mesenchymal stem) are multipotent stem cells with self-replication and multi-differentiation potential. Adult bone marrow is currently the main source of MSC. Bone marrow mesenchymal stem cells must be obtained by bone marrow puncture, causing some pain to the donor, and the proliferation and differentiation potential of bone marrow mesenchymal stem cells decrease with the age of the donor. Finding new sources of MSC has become a hot spot in stem cell research at home and abroad. In this study, MSCs were isolated from the umbilical cord of full-term fetus. The biological, immunological and antigraft versus host disease (acute graft versus host disease, aGVHD) effects of MSCs were systematically studied in order to find a new source of MSC. In the first part of the experiment, we established a method to isolate MSC from umbilical cord and compared with adult bone marrow derived MSC to study the biological characteristics of umbilical cord derived MSC. The results showed that the isolation rate of umbilical cord derived MSC was 100%, compared with bone marrow derived MSC, the proportion of umbilical cord derived MSC fibroblast colony forming unit (CFU-F) was higher than that of bone marrow derived MSC. The expression of HLA-I and CD106 in MSCs was lower than that in MSC derived from bone marrow (P0.05). Umbilical cord derived MSC morphology, most molecular phenotypes (CD13C CD29, CD44, CD105, CD73, CD166 positive, CD14, CD34, CD38 and CD45 negative, CD31 negative), cell cycle state (more than 80% of cells in G_0/G_1 phase), adipose and osteogenic differentiation ability, The ability of cytokine secretion (expression of IL-6 TPO-FLIL-6 and IL-3) and long-term hematopoietic support was similar to that of bone marrow-derived MSC. The second part of this experiment was to study the immunological characteristics of umbilical cord-derived MSC. Immunophenotypic analysis showed that MSC from umbilical cord source expressed HLA-I, did not express HLA-DRR, and did not express co-stimulatory molecules CD80 CD86 and CD40.3H-thymidine (3H-Tdr) incorporation assay. The results showed that cord derived MSC did not stimulate lymphocyte proliferation in mice. In addition, the mixed lymphocyte reaction in mice was significantly inhibited. In the third part of the experiment, the effect of umbilical cord MSC on aGVHD was studied by GVHD mouse model. The pathological changes of GVHD target organs (liver, small intestine and skin), lymphocyte subsets and cytokine levels in GVHD mice were studied. The results showed that umbilical cord derived MSC intravenous infusion could significantly reduce the severity of aGVHD in mice model of haploidentical transplantation. Its mechanism may be related to the increase of Th2 cytokines.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R329.2

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