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Application of ex vivo expanded hematopoietic stem cells stimulated with human umbilical venous endothelial cells for transplantation in children

人脐静脉内皮细胞刺激离体扩增造血干细胞在儿童移植中的应用

基本信息

批准号：
15591115
负责人：
FUJITA Ichiro
金额：
$ 1.92万
依托单位：
Saga University (2004)佐賀医科大学 (2003)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

The aim of this study was to establish the ex vivo expansion of hematopoietic stem cells by co-culture system with human umbilical venous endothelial cells(HUVEC). After the establishment of the co-culture system with HUVEC, hematopoietic stem cells continuously grew and proliferated. However, several problems including how to maintain the function of endothelial cells for a long time, or how to make an attachment of stem cells effectively with endothelial cells via membrane filter.The aim of the next study was to clarify effects of INF-γ on monocytes and macrophages, using U937 monocytic cells. We investigated expressions of inflammation-related proteins such as MCP-1 (Monocyte chemoattractant protein-1,COX-2 (Cyclooxyganase-2), iNOS (inducible nitric oxide synthetase), Ferritin, CRP(C-reactive protein), AngiotensinII receptor-1(AT1R), Scavenger receptor type A(SRA) and Oxidized LDL receptor-1(LOX-1) with fluorescent immnocytochemical staining. Stimulation with INF-γ for 24 hours caused upregulation of all these proteins in U937 cells strongly. The fluorescent intensity of these proteins was increased by more than 150% of that in unstimulated cells. In comparison with TNF-α, which is one of proinflammatory cytokines, TNF-α did not give such striking effect on U937 cells. Moreover, INF-γ seemed to accelerate uptake of Dil labeled oxidized LDL and induced morphological changes like foam cells in some cells. These results demonstrate that IFN-γ could activate monocytes strongly and variously, and that effects of FN-γ could play important roles in disease such as hemophagocytic syndrome, macrophage activating syndrome and atherosclerosis.

本研究旨在建立人脐静脉内皮细胞（HUVEC）体外培养造血干细胞的方法。与HUVEC共培养体系建立后，造血干细胞持续生长增殖。然而，如何长期维持内皮细胞的功能，以及如何通过膜滤膜使干细胞与内皮细胞有效地附着在一起，是目前研究的几个问题。下一项研究的目的是利用U937单核细胞阐明INF-γ对单核细胞和巨噬细胞的影响。我们用荧光免疫细胞化学染色研究了炎症相关蛋白如MCP-1（单核细胞趋化蛋白-1）、COX-2（环氧合酶-2）、iNOS（诱导型一氧化氮合成酶）、铁蛋白、CRP（c反应蛋白）、血管紧张素受体-1（AT1R）、清除率受体A型（SRA）和氧化LDL受体-1（LOX-1）的表达。用INF-γ刺激24小时可引起U937细胞中所有这些蛋白的强烈上调。这些蛋白的荧光强度比未受刺激的细胞增加了150%以上。与促炎因子之一TNF-α相比，TNF-α对U937细胞没有如此显著的作用。此外，INF-γ似乎加速了Dil标记的氧化LDL的摄取，并在一些细胞中诱导了泡沫细胞等形态变化。这些结果表明，IFN-γ可以强而多样地激活单核细胞，FN-γ在噬血细胞综合征、巨噬细胞活化综合征和动脉粥样硬化等疾病中发挥重要作用。