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

本研究旨在通过与人脐静脉内皮细胞(HUVEC)共培养体系建立造血干细胞的体外扩增体系。与人脐静脉内皮细胞共培养体系建立后，造血干细胞不断生长增殖。然而，如何长期维持内皮细胞的功能，以及如何通过膜过滤器将干细胞有效地附着到内皮细胞上，仍存在一些问题。下一步的研究目的是以U937单核细胞为研究对象，阐明干扰素-γ对单核细胞和巨噬细胞的影响。用荧光免疫细胞化学方法检测炎症相关蛋白MCP-1、COX-2、iNOS、铁蛋白、C反应蛋白、血管紧张素Ⅱ受体-1(AT1R)、清道夫受体A(SRA)和氧化低密度脂蛋白受体-1(LOX-1)的表达。经干扰素-γ作用24小时后，U937细胞中上述蛋白的表达均明显上调。这些蛋白质的荧光强度比未刺激的细胞增加了150%以上。与促炎细胞因子之一的肿瘤坏死因子-α相比，肿瘤坏死因子-α对U937细胞的作用不明显。此外，干扰素-γ似乎加速了DIL标记的氧化型低密度脂蛋白的摄取，并在一些细胞中诱导了类似泡沫细胞的形态变化。结果表明，干扰素-γ对单核细胞具有较强的激活作用，Fn-γ可能在吞噬血细胞综合征、巨噬细胞激活综合征和动脉粥样硬化等疾病中发挥重要作用。

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.