Inhibitory Effect Of ABO Blood Group Antibody On Erythroid Differentiation Of K562 Cells

ObjectiveTo reserch the inhibitory effection of ABO blood group antibody on erythroid differentiation of induced K562 cells, and to explore the interfered mechanism of mismatched ABO blood group antibody in erythroid differentiation and maturation process, as well as the influence on the expression of ABO blood group antigen.To study on the humoral immunity mechanism of pure red cell aplasia from cell experiment.Methods1. K562 cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum.2. To treat K562 cells with hemin in different concentration and to induce K562 cells differentiate into erythron.The maturity of K562 cells induced into erythroid differentiation was investigated by detecting the synthesis of hemoglobin with Benzidine staining.3. Detection of blood group antigen:the ABO blood group antigens on the surface of induced K562 cells were detected with absorption and elution test and micro lymphocytotoxicity test.4. the ABO blood type of K562 cell line was confirmed by sequencing.5. The inhibitory rate of K562 cells proliferation under the effect of different anti-H concentration was detected with MTT.6. DNA-free total RNA was isolated from induced K562 cells in different concentration of anti-H antibody.7. Applying SYBR GREEN real time PCR to investigate the relative FUT1 gene expression on induced K562 cells which was mediated in different concentration of anti-H antibody. 8. Cell apoptosis was detected by Annexin V/PI on induced K562 cells which was effected with different concentration of anti-H antibody.Results1. The K562 cells induced by different concentration of hemin in different days showed that the synthesis efficiency of hemoglobin under the 50uM/L of hemin for 4 days was the highest.2. As the low expression of blood type antigens on introduced K562 cells, the ABO blood group can't be detected by absorption and elution test and micro-lymphocytotoxicity test. The ABO blood type of K562 cell line was confirmed as 0 blood type by sequencing.3. The proliferative inhibitory rate of induced K562 cells showed statistical significance between 1:8 and 1:16 dilution group of anti-H antibody by MTT test. When anti-H dilution was below or equal to 1:8, there had evident inhibitory effect on the cell proliferation.4. The expression of FUT1 gene showed significant change under the different anti-H dilution in different culture days with the real time PCR test.When anti-H dilution was below or equal to 1:16, there had evident inhibitory effect on the expression of Futl gene.5. It showed that the anti-H could urge the K562 cells,which were induced into erythroid differentiation into apoptosis. The cell apoptosis rate was dramatic decline with the dilution of anti-H, and when the dilution of anti-H was over 1:16,the apoptosis rate didn't show significantly different.Conclusion1. The effectiveness of K562 cell induced into erythroid differentiation was different in different concentration of hemin.2. ABO blood group antibody could inhibit the proliferation of K562 cells induced into erythroid differentiation. The expressiong of hemoglobin inside the cells was obviously increased when the K562 cells were induced into erythron.but that of ABO blood group antigen on cell surface is very low. 3. The expression of FUT1 gene increased during the K562 cell differentiated into erythron, but anti-H among certain level could inhibit the gene expression.4. In certain concentration range, the ABO blood group antibody could inhibit erythroid differentiation and matrurate of induced K562 cells through cell apoptosis. From the cell test,we could infer that ABO blood group antibody could inhibit erythroid differentiation and matrurate of stem cell and maybe play an important role in the development of PRCA.5. The method of real time PCR we established could be used to detect the change of FUT1 gene expression, which also could be used to investigate the expression of the ABO blood group gene.6. The study provided a new thinking for researching on the mechanism of PRCA after transplantation of ABO incompatibility allogeneic haematopoietic stem cell.