The Function Of Insulin Receptor In Erythropoiesis

Mature red blood cells in the human body maintain the normal operation of various tissues and organs in the body by transporting oxygen and carbon dioxide.Clinical blood transfusion is one of the important means for the treatment of anemia patients,but the shortage of blood resources has become a common phenomenon.Studies have shown that insulin(INS)needs to be added during the differentiation of hematopoietic stem cells into erythrocytes,and INS can promote the colony forming unit(BFU-E)of early erythroid progenitor cells and the colony forming unit of late erythroid progenitor cells.(Colony forming unit-erythroid,CFU-E)proliferation,and play an anti-apoptotic effect.INS mediates cell surface signal transduction through insulin receptor(IR),and IR can also bind to RNA polymerase Ⅱ in the promoter region of INS function-related genes in the nucleus,thereby participating in the regulation of gene expression.Studies have shown that IR-mediated signaling is very important for pluripotency and lineage differentiation of pluripotent stem cells,and knockdown of IR gene expression in pluripotent stem cells can change the direction of cell differentiation.Erythropoiesis is a complex multi-stage process,which is divided into three stages:the early stage of erythropoiesis,the terminal stage of erythropoiesis and the maturation of reticulocytes,and IR is continuously expressed at each stage of erythropoiesis.To explore the effect of IR on the early stage of erythropoiesis,we added the insulin receptor inhibitor AGL-2263 to erythroid-differentiated D2 from cord blood(CB)-derived CD34+ cells.Inhibition,in order to explore the reasons for the inhibition of cell proliferation,we found that apoptosis was increased,colonyforming ability was decreased,and the cell cycle was arrested in the G0-G1 phase,and we speculated that these changes were responsible for the inhibition of cell proliferation.More importantly,we found that insulin receptor inhibitors blocked the differentiation process of CFU-E cells to GPA+ cells.It was found by cell slide staining that from D11,non-erythroid cells appeared in the drug-added group,and we speculated that insulin was affected by insulin.Body inhibitors block the erythroid differentiation process and lead to abnormal differentiation.To explore the effect of IR on the terminal stages of erythropoiesis,we added AGL-2263 to CB-derived CD34+cells to erythroid-differentiated D7.It was found that increased apoptosis and cell cycle arrest resulted in the inhibition of cell proliferation after dosing.We also found that the addition of AGL-2263 delayed the terminal differentiation of erythrocytes and resulted in abnormal erythrocyte differentiation.In order to confirm that AGL-2263 causes abnormal erythroid differentiation,we used flow cytometry to sort GPA+ cells on D7,cultured cells in erythroid culture system and full line culture system,and added AGL-2263.It was found that GPA-cells appeared in both erythroid and Mix culture systems,so we confirmed that AGL-2263 can cause abnormal differentiation of human erythrocytes.To explore the effect of IR on the denucleation of metaplastic erythrocytes,we added AGL-2263 to erythroid D13 from CB-derived CD34+cells,and found that AGL-2263 reduced the denucleation rate of metaplastic erythrocytes.In order to explore the mechanism by which insulin receptor inhibitors affect the terminal differentiation of erythrocytes,we performed RNA-seq detection.The KEGG results showed that compared with the control group,the hematopoietic lineage differentiation changed most significantly in the drug-added group.We analyzed key genes and found that the expression levels of these non-erythroid cell marker genes CD 14,TNF,HLA-DQB1,HLA-DRA,HLA-DRB1,HLA-DRB5,etc.were significantly up-regulated in the cells of the drug-added group,CSF1,The expression of CSF1R,CSF2RA and other key genes related to the regulation of cell differentiation were also significantly up-regulated in the cells of the drug-added group.We speculated that this may be the reason for the abnormal differentiation of the cells in the drug-added group.Through this project,we will explore the signal regulation mechanism involved in the regulation of erythroid differentiation by insulin receptors,which will be of great significance to the research on the process and regulation mechanism of erythropoiesis.It can provide a theoretical basis for the mass production of erythrocytes in vitro.