The Expressions Of G-CSFR、SCFR And Post Receptor Phosphorylated STAT5in CD34+CD59-and CD34+CD59+Bone Marrow Cells Of The Patients With Paroxysmal Nocturnal Hemoglobinuria

Objective:1. To measure the expressions of granulocyte colony-stimulating factor receptor (G-CSFR,CD114) and stem cell factor receptor (C-KIT,CD117) on the membrane of CD34+CD59-and CD34+CD59+bone marrow cells of the patients with paroxysmal nocturnal hemoglobinuria(PNH),and signaling pathway protein STAT5within the cytoplasm of those cells.2.To study the expressions of phosphorylated STAT5before and after invitro G-CSF or SCF stimulation,and evaluate the functions of G-CSF and SCF receptors on PNH clone cells.Methods:1. The expressions of CD114and CD117on the cell membrane and STAT5protein within the cytoplasm of bone marrow CD34+CD59+and CD34+CD59-cells from26PNH patients and14normal controls were examined by flow cytometry(FCM).2. Bone marrow mononuclear cells(BMMNC)of26PNH patients and14normal controls were stimulated in vitro with G-CSF(100ng/ml)or SCF(100ng/ml) for10minutes. Before and after these stimulations,the mean fluorescence intensity (MFI) of phosphorylated STAT5(P-STAT5) in CD34+CD59+BMMNC and CD34+CD59-BMMNC were measured by FCM.Results:1.①The percentage of CD114positive cells in CD34+CD59-cells of PNH patients was (43.23±19.77)%,which was significantly lower than that in CD34+CD59+cells of PNH patients(73.72±17.42)(P<0.01) or normal controls (65.91±13.70)(P<0.01). There was no statistic difference between the two latters(P>0.05);②The percentage of CD117positive cells in CD34+CD59-cells of PNH patients was (49.20±26.80)%,which was significantly lower than that in CD34+CD59+cells of PNH patients(67.62±17.41)(P<0.01) or normal controls (70.21±12.68)(P<0.01). There was no statistic difference between the two latters(P>0.05).2. The STAT5MFI in CD34+CD59-and CD34+CD59+cells of PNH patients and CD34+CD59+cells of normal controls was (270.01±181.26),(205.05±146.16), (227.39±156.65) respectively. There was no statistic difference among the three groups(P>0.05).3.①The P-STAT5MFI in unstimulated CD34+CD59-cells of PNH patients was (23.91±18.50),which was significantly lower than that in unstimulated CD34+CD59+cells of PNH patients(63.72±49.48)(P<0.01) or normal controls (61.20.±33.34)(P<0.01). There was no statistic difference between the two latters(P>0.05);②The P-STAT5MFI in G-CSF stimulated CD34+CD59-cells of PNH patients was (36.26±35.15),which was significantly lower than that in G-CSF stimulated CD34+CD59+cells of PNH patients (123.61±83.53)(P<0.01) or normal controls (115.69±58.64)(P<0.01), there was no statistic difference between the two latters(P>0.05);③The P-STAT5MFI in SCF stimulated CD34+CD59-cells of PNH patients was (33.59±27.44),which was significantly lower than that in SCF stimulated CD34+CD59+cells of PNH patients(123.54±97.04)(P<0.01) or normal controls (128.04±62.29)(P<0.01), there was no statistic difference between the two latters(P>0.05);④The increased P-STAT5MFI in G-CSF stimulated CD34+CD59-cells of PNH patients was (13.17±26.46),which was significantly lower than that in G-CSF stimulated CD34+CD59+cells of PNH patients(59.91±47.40)(P<0.01) or normal controls(54.49±51.54)(P<0.01). There was no statistic difference between the two latters(P>0.05);⑤The increased P-STAT5MFI in SCF stimulated CD34+CD59-cells of PNH patients was (9.77±17.10),which was significantly lower than that in SCF stimulated CD34+CD59+cells of PNH patients(59.83±66.79)(P<0.01) or normal controls(66.84±54.74)(P<0.01).There was no statistic difference between the two latters(P>0.05).Conclusions:1.In PNH, the expressions of CD114and CD117on bone marrow PNH clone cells were lower than that on normal clone cells, but the expression of signaling pathway protein STAT5within the cytoplasm was normal; 2. The expression of P-STAT5in G-CSF or SCF stimulated PNH clone cells was lower than that in normal clone cells. It indicated that CD114and CD117on PNH clone cells were lower functional.Therefore,we can apply the mechanisms that PNH clone cells responsed poorly to stimulation of cytokines,such as G-CSF and SCF in the chemotherapy of PNH and promote the proliferation of normal clone in stead of PNH clone.