OBJECTIVETo explore the influence of GATA-1 on expression of EpoR in bone marrow CD71+ cells of rat model with high altitude polycythemia (HAPC).

METHODSForty-eight male SD rats were randomly divided into normal control and HAPC model group. HAPC model was established at the altitude of 4 300 meters in the natural environment, and verified by bone marrow cell counts and hematological parameters. Myeloid CD71+ cells were separated by the density gradient centrifugation combined with magnetic activated cell sorting. The expression of EpoR on cell membrane was detected by flow cytometry and cell immunofluorescence. The expression changes of GATA-1 and EpoR mRNA and protein were detected by Q-PCR and Western blot, respectively. CD71+ cells were cultured under normoxia and hypoxia, respectively. After transfection for 96 h, the optimal interference sequence GATA-1 shRNA1 was selected. And the mRNA and protein expression level of GATA-1 and EpoR were detected by Q-PCR and Western blot respectively.

RESULTSThe animal model with HAPC was established successfully and comfirmed by the bone marrow cell counting and the hematologic parameters in comparison with that of the normal control. EpoR expression on the myeloid CD71+ cell membrane in HAPC group was significantly higher than that in normal control (P<0.05). The expression of GATA-1 and EpoR in myeloid CD71+ cells of HAPC group was higher than that in control group (P<0.05). The mRNA and protein expression of GATA-1 and EpoR in two groups positively correlated (control group, r=0.929, P<0.01, r=0.802, P<0.05; HAPC group, r=0.822, P<0.05, r=0.839, P<0.01). However, the mRNA and protein expression of EpoR at normoxia and hypoxia was significantly lower than that in negative control group after interfernce with GATA-1 shRNA1 for 96 h (P<0.05). And the expression of GATA-1 and EpoR under hypoxia was higher than that in normoxia.

CONCLUSIONThe effect of GATA-1 on EpoR expression may be correlated with the pathogenesis of HAPC.

Altitude ; Animals ; Antigens, CD ; metabolism ; Bone Marrow Cells ; metabolism ; Cell Separation ; Disease Models, Animal ; Flow Cytometry ; GATA1 Transcription Factor ; metabolism ; Male ; Polycythemia ; metabolism ; RNA, Messenger ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, Erythropoietin ; metabolism ; Receptors, Transferrin ; metabolism

OBJECTIVE: To explore the relationship between injury age and expressions of erythropoietin (EPO) and its receptor EPOR in the brain tissue of rats after cerebral injury. METHODS: Seventy-two rats were randomly divided into control group (36 rats) and cerebral injury group (36 rats). The rats were sacrificed at 1, 2, 4, 8, 12, 24 h after cerebral injury (6 rats at each time point) and the brain tissues were extracted. The expressions of mRNA and protein of EPO and EPOR at different time points were detected by real-time fluorescent quantitative PCR and Western bloting. RESULTS: The expressions of EPO and EPOR increased within 24 h after injury. The expressions of mRNA and protein of EPO were related to the injury age, and the correlations were 0.875, 0.911, respectively (P < 0.05). The expressions of mRNA and protein of EPOR were related to the injury age, and the correlation coefficients were 0.936, 0.905, respectively (P < 0.05). CONCLUSION The expressions of EPO and EPOR increase gradually in the early stage of the rat's cerebral injury, which are associated with the injury age and could be a useful value for estimating injury age.
Animals ; Brain/metabolism* ; Brain Injuries/pathology* ; Erythropoietin/metabolism* ; RNA, Messenger/metabolism* ; Random Allocation ; Rats ; Receptors, Erythropoietin/metabolism* ; Time Factors

This study was conducted to investigate the effects of erythropoietin (Epo) on both acute and chronic limb ischemia (ALI and CLI) and to evaluate the differences in mechanisms according to the method of Epo administration. Hindlimb ischemia was made in BALB/c mice with femoral artery ligation. The mice were divided into four groups: Group 1 (control, no treatment), Group 2 (ALI, early multiple doses), Group 3 (ALI, early single high dose), Group 4 (CLI, late multiple doses). Blood flow ratio significantly increased in Group 2 in 4 weeks. Expression of pAkt and Erythropoietin receptor were significantly higher in Group 2 on postoperative day (POD) 7. The number of CD31- and vascular endothelial growth factor-positive cells were significantly higher in Group 2 on POD 7 and 56. Group 3 and 4 showed a tendency of higher cell counts than the control. The early sustained Epo was effective in improving blood flow through angiogenesis. In chronic phase, weekly multiple dosing of Epo induced angiogenesis, however, the blood flow ratio did not increase significantly. The results of this study suggest that Epo administration during the acute phase followed by maintenance for several days may be important for increasing blood flow and angiogenesis.
Acute Disease ; Animals ; Chronic Disease ; Erythropoietin/*pharmacology ; Hindlimb/*blood supply ; Ischemia/*metabolism/pathology ; Laser-Doppler Flowmetry ; Male ; Mice ; Mice, Inbred BALB C ; Neovascularization, Physiologic/drug effects ; Proto-Oncogene Proteins c-akt/metabolism ; Receptors, Erythropoietin/metabolism ; Recombinant Proteins/pharmacology ; Vascular Endothelial Growth Factor A/metabolism

OBJECTIVEErythropoietin and erythropoietin receptor (EPO-R) are expressed in many kinds of tumors. The EPO/EPO-R signaling is involved in tumor cell proliferation, invasion and angiogenesis. The aim of this study was to detect the expression of EPO-R in non-small cell lung cancer (NSCLC), and explore its correlation with angiogenesis.

METHODSThe expression patterns of EPO and EPO-R in 31 cases of NSCLC tissues were detected by immunohistochemistry, and that in benign lung lesions of 21 patients as control. To analyze the correlation of EPO/EPO-R expression patterns and clinicopathological factors. CD34 was used to label the vascular endothelial cells and calculate the microvessel density (MVD).

RESULTSThe positive rates of EPO and EPO-R expression in NSCLC were 67.7% and 96.8%, respectively, significantly higher than those in the control ones. The positive rates of EPO and EPO-R expression in adjacent tissues were 19.4% and 35.5%, and in benign lesions were 9.5% and 19.0%, respectively (P < 0.001). The expression patterns of EPO/EPO-R were not related with pTNM stage, histological type, histological grade and lymph node metastasis (P > 0.05). Increased MVD was correlated with poor differentiation, lymph node metastasis, and advanced stage.

CONCLUSIONSHigh expression of EPO/EPO-R in NSCLC patients suggest that they may be involved in tumorigenesis. EPO/EPO-R expression and MVD are closely related, and they might be an endogenous stimulant of angiogenesis during the progression of non-small cell lung cancer. It may provide evidence for clinical diagnosis.

Antigens, CD34 ; metabolism ; Carcinoma, Non-Small-Cell Lung ; blood supply ; metabolism ; pathology ; Erythropoietin ; metabolism ; Humans ; Lung Neoplasms ; blood supply ; metabolism ; pathology ; Lymphatic Metastasis ; Microvessels ; pathology ; Neoplasm Staging ; Neovascularization, Pathologic ; Receptors, Erythropoietin ; metabolism

OBJECTIVETo study the expressions of erythropoietin receptor (EPOR) and thrombopoietin receptor (TPOR) on CD34+ CD59- and CD34+ CD59+ bone marrow (BM) cells from patients with paroxysmal nocturnal hemoglobinuria (PNH).

METHODS(1) The expressions of EPOR and TPOR on CD34+ CD59- and CD34+ CD59- BM cells from 26 PNH patients and 16 normal controls were examined by flow cytometry (FCM). (2) The mRNA expression of the EPOR and the TPOR in BM mononuclear cells (BMMNC) from 25 PNH patients and 13 normal controls were examined by RT-PCR.

RESULTS(1) The percentage of EPOR positive cells in PNH CD34+ CD59+ BMMNC [(30.67 +/- 18.30)%] was significantly higher than that in PNH CD34+ CD59- BMMNC [(8.05 +/- 3.51)%] (P < 0.01) and than that in control CD34+ CD59+ BMMNC [(8.24 +/- 6.51)%] (P < 0.01), but there was no obvious difference between the CD34+ CD59-BMMNC in PNH and CD34+ CD59+ BMMNC in control. (2) The percentage of TPOR positive cells in PNH CD34+ CD59+ BMMNC [(28.15 +/- 17.75)%] was significantly higher than that in PNH CD34+ CD59-BMMNC [(15.65 +/- 14.45)%] (P < 0.05) and than that in control CD34+ CD59+ BMMNC [(10.77 +/- .39)%] (P < 0.01), but there was no obvious difference between the CD34+ CD59- BMMNC in PNH and CD34+ CD59+ BMMNC in control. (3) There was no statistic difference in EPOR mRNA and TPOR mRNA expressions in BMMNCs between PNH patients group [(0.41 +/- 0.37) and (0.32 +/- 0.19), respectively] and control group [(0.47 +/- 0.33) and (0.40 +/- 0.29), respectively].

CONCLUSIONThe expression of EPOR and TPOR of PNH patients on BM CD34+ CD59+ cells are significantly higher than those on BM CD34+ CD59- cells. The difference may be due to abnormal transcription of both receptor coding genes.

Adult ; Bone Marrow Cells ; metabolism ; CD59 Antigens ; metabolism ; Case-Control Studies ; Cells, Cultured ; Female ; Flow Cytometry ; Hemoglobinuria, Paroxysmal ; metabolism ; Humans ; Male ; Middle Aged ; Receptors, Erythropoietin ; metabolism ; Receptors, Thrombopoietin ; metabolism ; Young Adult

OBJECTIVETo study the effects of hermap gene on kinases in erythroid signal transduction pathway and investigate the mechanism of hermap on erythroid differentiation.

METHODSThe K562 cells expressing hermap and hermap-siRNA respectively were established for up- and down-regulating the expression of hermap gene. These K562 cells were then induced by Ara-C to erythroid differentiation and analyzed at 0, 24, 48, 72 and 96 h, respectively, for cell morphology and biphenylamine staining positive cells, determination of CD235a, CD36, kinases p-STAT5, p-Akt, p-MAPK and p-c-JUN by FCM; and quantification of hermap gene and γ (Aγ,Gγ) globin gene by FQ-PCR.

RESULTSWith up-regulating hermap gene and inducing by Ara-C, K562 cells were changing to low ratio of nucleus to cytoplasm, cytoplasm colour from basophilic to pinkish or amethyst tinge, increase of number of biphenylamine positive cells and expression of CD235a, CD36, γ (Aγ,Gγ) globin gene, hermap gene and p-STAT5 from 0 to 96 h. At 0, 24, 48, 72 and 96 h of culture, the positive rates of p-STAT5 cells were detected of 0.46%, 4.54%, 20.01%, 23.65% and 33.08%, respectively. This results demonstrated that there was a positive correlation between expression of p-STAT5 and hermap gene expression (P < 0.05).

CONCLUSIONhermap gene can stimulate erythroid differentiation of Ara-C induced K562 cells mainly through JAK/STAT5 signal transduction pathway.

Cell Differentiation ; Erythrocyte Membrane ; Erythrocytes ; cytology ; Erythropoiesis ; Gene Expression ; Humans ; K562 Cells ; Receptors, Erythropoietin ; genetics ; STAT5 Transcription Factor ; metabolism ; Signal Transduction

OBJECTIVETo study the expression of erythropoietin (EPO) and its receptor (EPOR) in the brain of newborn rats suffering fetal distress.

METHODSA model of fetal distress was prepared by ligating bilateral uterine arteries of the rats with full-term pregnancy for 10 minutes before cesarean sections. The expression levels of EPO and EPOR in the brain of newborn rats were detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot at 0, 2, 6, 12, 24, 48, 72 hrs and 7 days after birth. Serum EPO levels were measured using ELISA simultaneously. The newborn rats born by cesarean sections which were not subjected to uterine artery ligation were used as the control group.

RESULTSThe expression of EPO protein and mRNA in brain tissues in the fetal distress group increased significantly compared with the control group 2, 6 and 12 hrs after birth (P<0.05). The expression of EPOR protein and mRNA in brain tissues in the fetal distress group increased significantly compared with the control group 2, 6, 12, 24 and 48 hrs, and 3 days after birth (P<0.05). Serum EPO levels in the fetal distress group were significantly higher than in the control group 2 hrs after birth.

CONCLUSIONSThe EPO and EPOR levels in the brain increase quickly after birth in newborn rats suffering from fetal distress. The EPOR is high expressed for a longer time than EPO. This can provide a basis for the treatment of neonatal brain damage induced by fetal distress by exogenous EPO.

Animals ; Animals, Newborn ; Brain ; metabolism ; pathology ; Erythropoietin ; blood ; genetics ; Female ; Fetal Distress ; metabolism ; Pregnancy ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Receptors, Erythropoietin ; blood ; genetics

OBJECTIVEOxygen toxicity is thought to be a major contributing factor in the pathogenesis of bronchopulmonary dysplasia (BPD). Animal experiments reveal that erythropoietin (EPO) may have protective effects against hyperoxic lung injury, but the mechanisms remain unknown. The aim of this study was to evaluate effects of hyperoxia on erythropoietin receptor expression in lung development of neonatal rats.

METHODSSeveral litters of Wistar pups were pooled together within 12 hours after birth and randomly divided into two groups (n = 24 in each): air-exposed control group and hyperoxia-exposed group. In hyperoxia-exposed group, the rats were exposed to 85% oxygen. Pups (n = 8) from each group were sacrificed on postnatal days 3, 7, and 14. The pulmonary histological and morphometric changes were observed after hematoxylin-eosin (HE) staining under light microscope. Radical alveolar counts (RAC) were compared between the two groups to evaluate the differences of alveolarization. Expressions of platelet endothelial cell adhesion molecule-1 (PECAM-1) and erythropoietin receptor (EPOR) in lung tissue were measured by immunohistochemistry. Expressions of EPOR mRNA and EPOR protein were measured by RT-PCR and Western blotting.

RESULTSIn hyperoxia-exposed group, there were a few inflammatory cells infiltration in interstitium on day 3 and inflammatory response worsened on day 7. Alveolar and capillary hypoplasia and interstitial fibrosis were evident on day 14. RAC increased in air-exposed control group along with the age in days. RAC decreased from day 7 in hyperoxia-exposed group compared with air-exposed control group [(6.85 ± 1.04) vs. (7.33 ± 1.0), P < 0.01], which was more evident on day 14 [(6.20 ± 1.58) vs. (9.07 ± 0.69), P < 0.001]. Expression of PECAM-1 protein increased in air-exposed control group along with the age in days. But in hyperoxia-exposed group, it decreased on day 7 and 14 [(15.14 ± 1.51) vs. (31.47 ± 2.43), (11.04 ± 1.76) vs. (41.41 ± 3.83), P < 0.001] compared with air-exposed control group. Expression of EPOR on day 3 in air-exposed control group was the strongest and weakened gradually with the increase of postnatal days. Expression of EPOR in hyperoxia-exposed group decreased on day 3 and became more evident on day 7 and day 14 compared with air-exposed control group [(1.62 ± 0.04) vs. (1.82 ± 0.06), P < 0.05; (0.48 ± 0.01) vs. (1.10 ± 0.07), (0.39 ± 0.04) vs. (0.87 ± 0.03), P < 0.001]. Expression of EPOR mRNA on day 3 in air-exposed control group was the strongest and was decreased significantly in hyperoxia-exposed group compared with air-exposed control group at all time points [(0.87 ± 0.07) vs. (1.1 ± 0.17), (0.18 ± 0.07) vs. (0.36 ± 0.08), P < 0.01;(0.14 ± 0.05) vs. (0.36 ± 0.09), P < 0.001].

CONCLUSIONSEPOR may participate in the modulation of normal lung development. Depressed expression of EPOR and PECAM-1 may be involved in the pathogenesis of alveolar and capillary hypoplasia induced by hyperoxia.

Animals ; Animals, Newborn ; Disease Models, Animal ; Female ; Lung Injury ; etiology ; metabolism ; Male ; Oxygen ; administration & dosage ; adverse effects ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism ; Pulmonary Alveoli ; drug effects ; metabolism ; Rats ; Rats, Wistar ; Receptors, Erythropoietin ; metabolism

This study was aimed to explore the expression of erythropoietin receptor (EPOR) on acute leukemia cells and its clinical significance. Bone marrow of 40 patients with acute leukemia (AL) and 24 patients with normal bone marrow as control group were collected. Samples came from outpatients and inpatients in our hospital. EPOR mRNA was detected by reverse transcription-PCR. The results showed that there was EPOR expression on AL cells, the expression rate was 57.5%, and the average expression level (Gray value) was 0.3549 ± 0.2800, but both were lower than that in control group (p < 0.05). There was no significant statistic difference of expression rate between acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL) (p > 0.05), and expression level of AML EPOR was higher than that of ALL (p < 0.05). It is concluded that there is EPOR expression on AL cells, while the expression rate and expression level are lower than those in control group (p < 0.05). There is no significant statistic difference of the expression rate between AML and ALL (p > 0.05), and the expression level of AML EPOR is higher than that of ALL (p < 0.05).
Case-Control Studies ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; RNA, Messenger ; genetics ; Receptors, Erythropoietin ; genetics ; metabolism

Anemia ; blood ; chemically induced ; drug therapy ; etiology ; therapy ; Antineoplastic Agents ; adverse effects ; Blood Transfusion ; Erythropoietin ; blood ; therapeutic use ; Hemoglobins ; metabolism ; Humans ; Neoplasms ; blood ; complications ; drug therapy ; Receptors, Erythropoietin ; blood