Effect Of Iron Overload On Hematopoiesis And Survival Of MDS And Its Mechanism

Objective: To study the effect of iron overload on hematopoietic function of MDS and its related mechanism,analyze the effect of iron overload on hematopoiesis and survival of patients with MDS,and further demonstrate the significance of iron overload in the development of MDS.Content: To construct an iron-overloaded MDS mouse model,to study the effect and mechanism of iron overload on bone marrow hematopoietic function and survival time of MDS mice,and to statistical clinical cases and analyze the effect of iron overload on bone marrow hematopoiesis and survival time of patients.Method:1.Establishment and identification of MDS iron overload mouse model: The exogenous mutant gene RUNX1-S291 fs was inserted into the bone marrow mononuclear cell genome by retrovirus and transplanted into C57BL/6 mice irradiated with 60 Co gamma rays.After 8 weeks,iron was injected intraperitoneally to establish an MDS iron overload mouse model.After 24 weeks of transplantation,the peripheral blood,bone marrow,femur,liver and spleen of mice were taken for identification: peripheral blood and bone marrow cells were stained by Wright’s staining to observe morphological features;femur,liver and spleen tissues were examined by HE staining to detect pathological changes;liver,spleen,and bone marrow Prussian blue staining was used to observe iron deposition.Flow cytometry was used to detect the expression of bone marrow cell antigen in mice.Bone marrow mononuclear cells and spleen proteins were subjected to Western blotting to confirm the expression of RUNX1-S291 fs gene.Post-transplant mice regularly monitored blood routine and transplant cell chimerism.2.Effects and mechanisms of iron overload on hematopoiesis in MDS mice: After 24 weeks of transplantation,bone marrow cells of mice were harvested,flow cytometry analysis of the frequency of bone marrow hematopoietic stem and progenitor cells in mice,the proportion of red blood cells in each phase,and the apoptosis of bone marrow cells;take mouse bone marrow cells for colony culture,repeated plating experiments,and competitive transplantation experiments to detect the function of mouse hematopoietic stem and progenitor cells;the level of reactive oxygen species in mouse bone marrow fluid and bone marrow erythrocytes,the expression level of reactive oxygen species production and clearance genes in mouse bone marrow cells,and m RNA expression levels of TGF-β family related genes were measured;the expression level of GDF11 protein was detected by ELISA;the survival time of each group of mice was observed and the end point was 360 days after transplantation.3.Effect of iron overload on hematopoiesis and survival of MDS patients: A total of 101 MDS patients from January 2008 to December 2017 in Tianjin First Central Hospital were counted to analyze the effect of iron overload on various types of bone marrow cells on smears;as well as the effect of iron overload on patient survival time,multivariate analysis was used to demonstrate the prognostic factors associated with MDS patients.Result:1.The pathological features of MDS and iron overload were detected in the RUNX1-S291 fs mutant mice injected with iron,and the MDS iron overload mouse model was successfully constructed to lay a foundation for studying the effect of iron overload on MDS.2.Iron overload reduced the number of normal hematopoietic stem and progenitor cells and affected red blood cell maturation in MDS mice.Iron overload affected red blood cell colony formation in normal HSPCs in MDS mice.3.Iron overload inhibits red blood cell hematopoiesis in MDS mice through active oxygen,and GDF11 plays a role in inducing reactive oxygen species production;4.Iron overload shortened the survival time of MDS mice,but no statistical difference was found in the end point of normal mice.5.In patients with low-to-moderate risk MDS,iron overload is one of the patients’ independent prognostic factors,and can reduce the number of early erythroid cells in patients and shorten the median survival of patients.Conclusions:In this study,MDS iron overload mouse model was successfully constructed by RUNX1-S291 fs gene and in vitro injection of iron.Our preliminary findings indicate that iron overload impairs the proportion and function of normal HSPCs in MDS mice,particularly in erythroid hematopoiesis,at least in part through an increase in ROS production induced by GDF11.Iron overload shortens the survival time of MDS mice.In patients with low-to-moderate risk MDS,iron overload is one of the patients’ independent prognostic factors,and can reduce the number of early erythroid cells in patients and shorten the median survival of patients.