The Mechanism About Erythrocytes Senescence And Unopsonized-uptaken

The life span of circulating red blood cells (RBCs) is about120days in humans, it is significant to prolong the life span of RBCs for clinic blood transfusion and make full use of blood resources. The unopsonized-uptaken process in which CD47-SIRP a signaling system involved plays an important role for erythrocytes aging and phagocytosis. Some valuable results about this system have been reported in these years. However, these important discoveries are mainly directed to mice erythrocytes. The main purpose of this article is to investigate erythrocyte membrane protein changes and the role of the signal pathway of CD47-SIRP a in human red blood cell aging and phagocytosis process.Firstly, we prepared red blood cells of different age by storing RBCs at4℃and37℃in vitro, and young RBCs and old RBCs from RBCs in vivo by capillary centrifugation, and then, we extracted the erythrocyte ghosts and vesicles. The expression of CD47, GPA on red blood cells membrane from4℃,37℃stored human red blood cells in vitro and in vivo and was detected by Western blot, the same means is also used to detect the expression of CD47, GPA on vesicles from4℃stored RBCs. Moreover, the flow cytometry was applied to measure the results about CD47, complement regulatory protein(CD35, CD55, CD59) on erythrocyte ghosts from4℃stored RBCs. The results showed that the CD47, GPA on RBCs’membrane stored4℃in vitro reduced significantly during the storage, the largest decline of CD47was82.64%(37℃in vitro84.51%; in vivo36.6%). Also, the flow cytometry got the same results. Moreover, we used Western blot to detect the expression of CD47, GPA on vesicles from4℃stored RBCs, and we found that the quantity of CD47on vesicles stored for40days was2times than volume of CD47on vesicle saved10days. These results show that, in the erythrocyte aging process, the erythrocyte membrane protein continuously reduced by vesicles form.Human monocytic leukemia (THP-1) cells cultured in vitro were used to sallow the IgG sensitized RBCs, young RBCs and old RBCs from capillary centrifugation and RBCs blocked with F(ab’)2fragments of anti-CD47monoclonal antibody. After Wright-Jim staining, we observed the result of englobement state. On the basis, we use the immune precipitation method to extract the total tyrosine phosphorylated proteins from THP-1which had swallowed RBCs of different age. Furthermore, we detected the volume of phosphorylated SIRP a by Western blot. The aim is to find the different among THP-1swallowed RBCs of different age.The monocyte monolayer analysis experimental discovered that THP-1is easier to swallow the old RBCs(35.32%) than the young RBCs(13.72%)(n=7,P<0.02); and THP-1is prone to swallow the blockade RBCs(31.51%) compared with fresh RBCs(2.22%)(n=7,P<0.02). Meanwhile, the level of SIRP a tyrosine phosphorylation on THP-1which had swallowed the different RBCs was also distinctive. Compared with the young RBCs phagocytosis, when THP-1swallowed the old RBCs, the amount of phosphorylated SIRP a declined57.12%. These results suggest that CD47-SIRPα inhibiting signal intensity decreased means the phagocytic signal enhanced and CD47-SIRP a signaling might be a key mechanism in controlling the clearance of senescence RBCs.In addition, in the red cell aging and phagocytic process, besides the presence of CD47-SIRP a signaling system, there is the opsonized phagocytosis process involved in complement and antibody. In order to explore the function of the complement regulatory protein in RBCs opsonized phagocytosis process, we primarily used Methoxy polyethylene glycol-NHS propionic acid ester (mPEG-SPA) and F(ab’)2fragments of antibody to block the complement regulatory protein. The results of Flow cytometry show that complement regulatory protein CD35, CD55, CD59had different decline, the largest decline of the expression of CRP in CD35was75.5%; CD55was66.93%; CD59was54.23%, so these proteins have some important functions during RBCs aged. In future, we need the more experiment to prove this hypothesis.