Mechanistic Studies On Deficiency Of Human RBCs In Hemato-lymphoid System Humanized Mice

ObjectiveHumanized mice is a kind of mouse model which carrying functional human gene,cell or tissue,is usually used to study human biology and pathology in vivo.Humanized mice with human hemato-lymphoid system is an important model.Using transgenic method or homologous recombination to display human gene in mouse could improve the composition of human hemato-lymphoid system and the function of human immune system,and enhance the availability of human disease simulation.Through the study and development in the past 30 years,hemato-lymphoid system humanized mice are becoming more perfect,has already been widely used in HIV,cancer,infectious disease,blood disease,and so on.However,the rigorous rejection of mouse innate immune system to human red blood cell hampers the human red blood cell reconstitution in humanized mice,this is one major short slab of the model.Thus,lucubrate the mechanism of human red blood cell rejection by mouse immune system and construct a novel humanized mouse model with high level red blood cell reconstitution is very important to human erythropoiesis and relevant disease.For this purpose,during this study,immunodeficient mice was used as study object,both in vivo and in vitro assays were used to elucidate the mechanism of human red blood cell rejection by immunodeficient mice phagocytic cells,and we used related reagent of this mechanism to construct humanized mice model with stable human red blood cell reconstitution.Methods(1)Phagocyte phagocytosis: we co-cultured human red blood cells with mouse phagocytes,then using Delta Vision Elite Imaging System to observe the phagocytosis.(2)Adherence of myeloid phagocytes in vitro: we used fluorescence microscope to observe the adherence of red blood cells to myeloid phagocytes from immunodeficient mice in different medium.Red blood cells including mouse red blood cells and human red blood cells,mouse myeloid phagocytes including macrophages,neutrophils and endothelial cells,medium including 1640 media,fresh human sera,fresh mouse sera,heat shock treated mouse sera(56 ?,30min),antibody neutralized mouse sera,cobra venom factor in vivo and in vitro treated mouse sera,and 1640 media added with mouse complement.And we then used Image-Pro plus software to analyze adherence ratio of red blood cells to myeloid phagocytes.(3)Red blood cells transfusion: we used flow cytometry to detect the clearance of human red blood cells in immunodeficient mice,the mice treated with clodronate,cobra venom factor,PBS,or clodronate combined with cobra venom factor.(4)Hemato-lymphoid system humanized mice construction: we injected human fetal liver cells into immunodeficient mice through caudal vein to construct human stem cell differentiation model,and used flow cytometry to detect human hematopoietic stem cell differentiation at different time points in cobra venom factor and clodronate treated mice model,including myeloid cell and red blood cell reconstitution proportion.(5)Human red blood cell morphology observation: we used Delta Vision Elite Imaging System to observe the morphology of human red blood cell in different micro-environment,including PBS,1640 medium,fresh human sera,fresh mouse sera,and mouse sera added with human sera.Result(1)Phagocytes of NOD/SCID mice will engulf human red blood cells in vitro.(2)Sera from immunodeficient mice,but not human,can promote the adherence of human red blood cells to mouse myeloid phagocytes,myeloid phagocytes including macrophages,neutrophils and endothelial cells,mousecomplement opsonization of human red blood cell is the key point of the adherence appearance.(3)Using cobra venom factor alone to consume mouse complement cannot improve the survival of transfused human red blood cells in immunodeficient mice,but can prolong the survival in immunodeficient mice with macrophages depletion.(4)Using cobra venom factor can improve human red blood cell reconstitution in immunodeficient mice with human hematopoietic stem cell transplantation whose macropahges has been depleted.(5)Human red blood cells become spinous and apoptotic in immunodeficient mice sera,human sera addition will recover the morphology and prohibit the adherence.ConclusionOur study provides direct evidence to prove that complement mediates human red blood cell rejection in immunodeficient mice,and improve human red blood cell differentiation in immunodeficient mice with human hematopoietic stem cell transplantation whose macrophages has been depleted at a certain degree,and provide a new orientation for preparing a better human red blood cell reconstitution in humanized mice.