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

本研究旨在建立造血干细胞与人脐静脉内皮细胞（HUVEC）共培养体系体外扩增造血干细胞。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，如何使干细胞通过膜过滤器有效地与内皮细胞贴附，仍是一个亟待解决的问题，本研究拟以U937单核细胞为研究对象，探讨INF-γ对单核细胞和巨噬细胞的影响。采用荧光免疫细胞化学染色法检测炎症相关蛋白MCP-1、考克斯-2、iNOS、铁蛋白、C反应蛋白、血管紧张素Ⅱ受体1、清道夫受体A和氧化低密度脂蛋白受体1的表达。INF-γ刺激24 h后，U937细胞中上述蛋白表达均明显上调。这些蛋白质的荧光强度增加了150%以上，在未受刺激的细胞。与促炎细胞因子TNF-α相比，TNF-α对U937细胞的作用不明显。INF-γ可促进Dil标记的氧化型LDL的摄取，并引起部分细胞出现泡沫细胞的形态学改变。这些结果表明，FN-γ可以强烈而多样地激活单核细胞，并且FN-γ的作用在噬血细胞综合征、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.