Effects Of The Hydrolysates Of EPO, Antibodies Of EPO Or EPOR On Erythroid Cells Proliferation And Differentiation Stimulatied By EPO

Objective: High altitude polycythemia (HAPC) is a kind of polycythemia caused by hypoxia in high altitude areas. The blood cell of HAPC patients would recover to normal level after they return to low altitude areas, which indicate the number of red blood cells of HAPC patients can be regulated. Erythropoietin (EPO) is the most important regulatory factor in the development, proliferation and differentiation of erythroid cells. EPO and EPO receptor (EPOR) are key factors in HAPC pathogenesis. THerefore, if we the generation of red blood cells was controled, HAPC might get effective treatment. In this study, we observed whether antibody of EPO, antibody of EPOR, and the polypeptide fragments (hydrolysates) from EPO enzymolysis can inhibit proliferation and differentiation of erythroid cells.Methods: In the first part of the experiments, adult SD rats, 150-160 g, were killed and sanitized, the thighbones were taken out. Then the marrow was washed out with the DMEM medium. CFU-E cells were cultured with methocel. The cells were incubated in 4 different conditions (4 groups): 1.normal control group: without EPO; 2. EPO group: stimulating by EPO; 3. EPO and EPO antibody group: with EPO and EPO antibody; 4. EPO and EPOR antibody group: with EPO and EPOR antibody.In the second part of the experiments, the cells were incubated in the following conditions (4 groups): 1. normal control group (with no EPO);2. EPO group (with EPO); 3. EPO and inhibitor-treated trypsin(ITT) group (with EPO and inhibitor-treated trypsin); 4. EPO and EPO hydrolysates group (with EPO and EPO hydrolysates). Hydrolysates were obtained through hydrolysis of EPO with trypsin and identified with SDS-PAGE. Cells were cultured for 7 days under the conditions of the saturation morsture, 37℃and 5%CO₂. Then the count of CFU-E colony forming units and the cell number of CFU-E in each colony forming unit were conducted. CFU-E cells were identified with microscope and the identification standards are that the cell number in each CFU-E colony forming unit should be equal to or larger than 8 and that the color of the cells should be reddish yellow.Results:1. There was a lot of CFU-E colony forming units in normal control group, but fewer colone forming units in the EPO and EPO antibody group. The difference between these two groups was greatly significant (p<0.01). The cell number in each CFU-E colony forming unit was large in the former group, while the corresponding value in the latter group is much smaller, which showed dramatically remarkable difference (p<0.01).2. The count of CFU-E colony forming units in EPO and EPOR antibody group was similar to that in normal control group. There existed no striking difference between these two groups (p>0.05).Besides, The cell number of the CFU-E colony forming units in EPO and EPOR antibody group was also similar to the corresponding value in normal control group (p>0.05).3. There was much fewer CFU-E colony forming units in EPO and EPO hydrolysates group than in normal control group (p<0.01). And the cell number in each CFU-E colony forming unit was much smaller in the former group than in the latter group, which also showed dramatically pronounced difference (p<0.01).Conclusion:1. EPO-antibody can hibit the proliferation and differentiation of the EPO, this may be a new approach for the treatment of HAPC.2. It may be deduced from the effects of the polypeptide fragments acquired in EPO hydrolization with trypsin that these fragments might be one of the drugs or pathways to treat HAPC.3. Antibody of EPOR has no effect on the proliferation and differentiation of EPO.