| Objective:1.To study the expressions of erythropoietin receptor (EPOR) and thrombopoietin receptor (TPOR) on bone marrow CD34+CD59- and CD34+CD59+ cells of the patients with paroxysmal nocturnal hemoglobinuria (PNH) and the STAT5 phosphorylation level in those cells after EPO or TPO stimulation in vitro. 2.To study the expressions of apoptosis receptor FAS and anti-apoptotic protein Bcl-2 on bone marrow CD34+CD59- and CD34+CD59+ cells of the patients with PNH.Methods:1. Bone marrow CD34+CD59+ cells and CD34+CD59- cells from 26 PNH patients and 16 normal controls were examined for the expressions of EPOR and TPOR on the cell membrane by flow-cytometry.2. Bone marrow mononuclear cells(BMMNC) from 25 PNH patients and 13 normal controls were examined for the mRNA expression of the EPOR and the TPOR by RT-PCR.3. BMMNC were extracted from 23 PNH patients and 11 normal controls. The mean fluorescence intensity (MFI) of phosphorylated STAT5 (P-STAT5) in CD34+CD59+ cells and CD34+CD59- cells,with or without the stimulation of 10U/ml EPO or 50U/ml TPO, were examined by flow-cytometry.4. Bone marrow CD34+CD59+ cells and CD34+CD59- cells from 25 PNH patients and 15 normal controls were examined for the expressions of apoptosis receptor FAS and anti-apoptotic protein Bcl-2 on the cell membrane by flow-cytometry.Results:1.①The percentage of EPOR positive cells in PNH CD34+CD59+ cells was (30.67±18.30)%,which was higher than that in PNH CD34+CD59- cells (8.05±3.51) %(p<0.01) and that in control CD34+CD59+ cells (8.24±6.51)%(p<0.01),but there was no obvious difference of EPOR expression between the CD34+CD59- cells in PNH and CD34+CD59+cells in control (p>0.05)②The percentage of TPOR positive cells in PNH CD34+CD59+ cells was (28.15±17.75)%,which was higher than that in PNH CD34+CD59- cells (15.65±14.45)% (p<0.05) and that in control CD34+CD59+cells (10.77±8.39)% (p<0.01), but there was no obvious difference of TPOR expression between the CD34+CD59-cells in PNH and CD34+CD59+cells in control (p>0.05)2.There was no obvious difference of EPORmRNA and TPORmRNA expressions between PNH BMMNC and control BMMNC (p>0.05)3.①Without stimulation, the P-STAT5 MFI in CD34+CD59-cells of PNH patients was (30.53±14.64),which was significantly lower than that of CD34+CD59+ cells (74.19±47.38) (p<0.01) and the normal controls CD34+CD59+ cells (59.39±23.48) (p <0.05), there was no statistic difference between the CD34+CD59+cells of PNH patients and the normal controls CD34+CD59+cells (p>0.05)②With the EPO stimulation, the P-STAT5 MFI in CD34+CD59-cells of PNH patients was (49.19±24.03),which was significantly lower than that of CD34+CD59+ cells (119.51±82.15) (p<0.01) and the normal controls CD34+CD59+cells (78.63±47.02) (p<0.05), there was no statistic difference between the CD34+CD59+ cells of PNH patients and the normal controls CD34+CD59+ cells (p>0.05)③With the TPO stimulation, the P-STAT5 MFI in CD34+CD59-cells of PNH patients was (51.15±41.24),which was significantly lower than that of CD34+CD59+ cells (123.75±86.90)(p<0.05) and the normal controls CD34+CD59+ cells (97.66±53.25) (p<0.05), there was no statistic difference between CD34+CD59+ cells of PNH patients and the normal controls CD34+CD59+cells (p>0.05)④There existed increased P-STAT5 MFI after EPO stimulation, the increased value of the CD34+CD59+cells in PNH patients was (49.12±10.61),which was significantly higher than that of CD34+CD59-cells (17.11±4.23) (p<0.01).④There existed increased P-STAT5 MFI after TPO stimulation, the increased value of the CD34+CD59+cells in PNH patients was (54.40±43.26),which was significantly higher than that of CD34+CD59-cells (16.21±5.79)(p<0.01).4.①The percentage of FAS positive cells in PNH CD34+CD59+ cells was (55.56±25.30)%,which was higher than that in PNH CD34+CD59- cells (33.34±29.28)%(p<0.01) and that in control CD34+CD59+ cells (33.45±18.68)% (p<0.01),but there was no obvious difference of FAS expression between the CD34+CD59- cells in PNH and CD34+CD59+ cells in control (p>0.05)②The percentage of Bcl-2 positive cells in PNH CD34+CD59+ cells was (56.67±17.67)%,which was higher than that in PNH CD34+CD59- cells (41.71±24.67)% (p<0.05),but there was no obvious difference of Bcl-2 expression between the CD34+CD59+cells (49.85±18.20)% in control and CD34+CD59+ cells, CD34+CD59-cells in PNH (p>0.05)Conclusions:1. In PNH, EPOR and TPOR expressions on bone marrow CD34+CD59- cells decreased obviously compared to those on CD34+CD59+ cells, and these decreases might not be due to abnormal transcription of both receptor coding genes. With or without the stimulation of EPO or TPO, the level of P-STAT5 in CD34+CD59+ cells of PNH patients were significantly higher than that of CD34+CD59-cells, indicating that the proliferation and differentiation of normal clones in PNH patients were better than those of abnormal clones after the administration of hematopoietic growth factors.2. In PNH, apoptosis receptor FAS and anti-apoptotic protein Bcl-2 expressions on bone marrow CD34+CD59- cells decreased obviously compared to those on CD34+CD59+ cells, indicating that the decreasing of the apoptosis receptor FAS expressions might enhanced the ability of anti-apoptoic in PNH clone cells,which were not involving the differences of Bcl-2 expressions. |
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