OBJECTIVE: To investigate the effect of Rheb1 in the development of mouse megakaryocyte-erythroid progenitor cells and its related mechanism. METHODS: Rheb1 was specifically knocked-out in the hematopoietic system of Vav1-Cre;Rheb1^fl/fl mice(Rheb1^Δ/Δ mice). Flow cytometry was used to detect the percentage of red blood cells in peripheral blood and erythroid cells in bone marrow in Vav1-Cre;Rheb1^fl/fl mice and control mice. The CFC assay was used to detect the differentiation ability of Rheb1 KO megakaryocyte-erythroid progenitor cells and control cells. Real-time fluorescence quantification PCR was used to detect the relative expression of PU.1,GATA-1,GATA-2,CEBPα and CEBPβ of Rheb1 KO megakaryocyte-erythroid progenitor cells and control cells. Rapamycin was added to the culture medium, and it was used to detect the changes in cloning ability of megakaryocyte-erythroid progenitor cells from wild-type mice in vitro. RESULTS: After Rheb1 was knocked out, the development and stress response ability of megakaryocyte-erythroid progenitor cells in mice were weaken and the differentiation ability of megakaryocyte-erythroid progenitor cells in vitro was weaken. Moreover, the expression of GATA-1 of megakaryocyte-erythroid progenitor cells was decreased. Further, rapamycin could inhibit the differentiative capacity of megakaryocyte-erythroid progenitor cells in vitro. CONCLUSION Rheb1 can regulate the development of megakaryocyte-erythroid progenitor cells probably through the mTOR signaling pathway in mice.
Animals ; Cell Differentiation ; Erythrocytes ; Flow Cytometry ; Megakaryocyte-Erythroid Progenitor Cells ; Megakaryocytes ; Mice ; Signal Transduction

OBJECTIVE: To investigate the changes of GATA-1 protein expression during erythroid differentiation of K562 cells under hypoxia and how GATA-1 can regulate erythroid differentiation by up-regulating the expression of miR-451a and inhibiting the expression of 14-3-3ζ. METHODS: K562 cells were divided into 2 groups: the normoxia group and the hypoxia group, after the induction of hemin for 96 h, the positive cells rate of the benzidine staining, the mRNA expression of γ-globin and the expression of CD235a were detected, and the success of the model was verified. The changes of GATA-1 and miR-451a expression in the above-mentioned 2 groups, the changes of miR-451a expression after over-expressed GATA-1 were detected by Western blot and qRT-PCR. The cells in normoxic group and hypoxia group were divided into negative control group (NC group) and miR-451a over-expression group respectively, and the degree of erythroid differentiation in the four groups was judged according to the corresponding erythroid differentiation indexes, and the expression of 14-3-3ζ was detected by Western blot after over-expressed miR-451a. RESULTS: The positive cell rate of benzidine staining, mRNA expression of γ-globin and the expression of CD235a after 96 h induction by K562 cells under hypoxia were significantly higher than 0 h, suggesting that the erythroid differentiation model of K562 cells under hypoxia was replicated successfully. The expression levels of GATA-1 protein and miR-451a in the hypoxic group were significantly higher than that in the normoxic group (P<0.05). The expression level of miR-451a in hypoxia group was significantly higher than that in NC group after overexpressed GATA-1 (P<0.05). After over-expressed of miR-451a under hypoxia, the positive cell rate of benzidine staining, the mRNA expression level of γ-globin and the expression of CD235a were significantly higher than those in NC group (P<0.05). The expression level of 14-3-3ζ protein in miR-451a over-expressed group was lower than that in NC group under hypoxia (P<0.05). CONCLUSION Hypoxia can significantly increase the expression of GATA-1 protein, and the increase of GATA-1 expression can up-regulate the expression of miR-451a, thereby inhibiting the expression of 14-3-3ζ protein, which hinders the cell proliferation in erythroid differentiation model of K562 cells and plays an important role in promoting erythroid differentiation.
14-3-3 Proteins ; Cell Differentiation ; Erythroid Cells/metabolism* ; GATA1 Transcription Factor/metabolism* ; Humans ; Hypoxia ; K562 Cells ; MicroRNAs/genetics*

OBJECTIVE: To compare the efficacy of directional erythroid differentiation in different serum free culture systems and to screen the optimal culture systems for inducing the differentiation of umbilical cord blood hematopoietic stem and progenior cells (HSPC) to erythroid cells. METHODS: The CD34 cells from umbilical blood munonuclear cells were sorted by using the magnetic beads, and were inoculated into 3 different of culture systems (system 1, 2 and 3 respectively), to induce erythrold differentiation by 3 stage culture. The living cells were counted in different differentiation stages and were observed by Wright-Giemsa staining; the expression of CD71 and CD235a on cell surface was detected by flow cytometry, the erythroid differentiation pteency was detected via colony-forming test. RESULTS: The ability of system 2 to promote the HSPC proliferation was the strongest, the efficacy of system 3 to promote the erythroid differentiation of HSPC was the most optimal; the proliferation ability of cells cultured in system 2 for 2-15 days all was higher than that of cells cutured in system 1 and 3 (P＜0.05). The flow cytometry detection showed that the expression of CD71 and CD235a on surface of cells cultured in system 3 was the highest, the CD235a percentage on day 15 of differentiation in system 3 was (92.33±3.89)%, that in system 2 was (84.67±3.12)%, while that in system 1 was (72.17±6.83)% (P＜0.05). Cell morplologic detection showed that throid differentiation was accelerated on day 12, the percentage of orthochromatic erythrocytes in system 3 was (67.67±2.08)% which was 10.69 and 25.34 times higher than that in system 2 and 1 respectively (P＜0.05). The colony-forming test showed the ratio of BFU-E in system 3 increased gradually on day 3-9 (r=0.99, P＜0.05), which was significanlly higher than that in system 2 and 1 on day 9 (90.35±5.52% vs 77.06±2.26% and 74.50±3.95%). CONCLUSION Culture system 3 is the most effective serum-free erythroid differentiation system, and the culture system 2 is the most powerful HSPC proliferation system. This study results provide a technical basis for further efficiently increasing and inducing the erythroid proliferation and differentiation of HSPC, and also provide culture system in vitro for the clinical application and basic research.
Antigens, CD34 ; Cell Differentiation ; Cells, Cultured ; Culture Media, Serum-Free ; Erythroid Precursor Cells ; Fetal Blood ; Humans

This study investigated the effects of N-acetylcysteine (NAC) and ascorbic acid (AA) on hemin-induced K562 cell erythroid differentiation and the role of reactive oxygen species (ROS) in this process. Hemin increased ROS levels in a concentration-dependent manner, whereas NAC and AA had opposite effects. Both NAC and AA eliminated transient increased ROS levels after hemin treatment, inhibited hemin-induced hemoglobin synthesis, and decreased mRNA expression levels of β-globin, γ-globin, and GATA-1 genes significantly. Pretreatment with 5,000 μmol/L AA for 2 h resulted in a considerably lower inhibition ratio of hemoglobin synthesis than that when pretreated for 24 h, whereas the ROS levels were the lowest when treated with 5,000 μmol/L AA for 2 h. These results show that NAC and AA might inhibit hemin-induced K562 cell erythroid differentiation by downregulating ROS levels.
Acetylcysteine ; pharmacology ; Antioxidants ; pharmacology ; Ascorbic Acid ; pharmacology ; Cell Differentiation ; drug effects ; Down-Regulation ; Erythroid Cells ; drug effects ; Hemin ; pharmacology ; Humans ; K562 Cells ; Reactive Oxygen Species ; metabolism

Objective: To understand the effect of sirolimus on the erythropoiesis of K562 cell line and bone marrow cells from pure red cell aplasia (PRCA) patients and normal controls. Methods: Different concentrations (10, 100, 1 000 nmol/L) of sirolimus were added to the K562 cell line or bone marrow cells from PRCA patients or normal controls and cultured 14 days for BFU-E formation. Meanwhile, sirolimus was also added to the serum treated PRCA bone marrow cells to cultivate for the same priod of time. Results: Neither K562 cells, bone marrow cells from PRCA patients or normal controls showed any difference when sirolimus was added to the culture system for BFU-E. However, BFU-E formation decreased after serum was added in PRCA patients (76.40±22.48 vs 136.33±12.58, t=-4.329, P=0.001) and this suppression of BFU-E was partly corrected by 1 000 nmol/L sirolimus treatment (97.14±15.83 vs 76.40±22.48, P=0.038). Conclusions: Sirolimus may modulate the suppression of erythropoiesis by serum instead of directly stimulate the growth of red blood cells in PRCA patients.
Erythroid Precursor Cells ; Erythropoiesis ; Humans ; K562 Cells ; Red-Cell Aplasia, Pure ; Sirolimus

BACKGROUND: Due to the tropism of human parvovirus B19 to erythroid progenitor cells, infection in patients with an underlying hemolytic disorder such as beta-thalassemia major leads to suppression of erythrocyte formation, referred to as transient aplasia crisis (TAC), which may be life-threatening. We investigated the prevalence of parvovirus B19 among patients with beta thalassemia major attending the Zafar Adult Thalassemia Clinic in Tehran, Iran. METHODS: This cross-sectional study was performed to determine the presence of parvovirus B19 DNA in blood samples and parvovirus B19 genotypes in plasma samples of patients with thalassemia major. The population consisted of 150 patients with beta-thalassemia major who attended the Zafar clinic in Tehran. Specimens were studied using a real-time polymerase chain reaction assay. RESULTS: The prevalence of parvovirus B19 in our study population was 4%. Of 150 patients with thalassemia, six (4%) were positive for B19 DNA. There was no significant correlation between blood transfusion frequency and B19 DNA positivity. Finally, phylogenetic analysis of human parvovirus B19 revealed genotype I in these six patients. CONCLUSION: In this study, acute B19 infections were detected in patients with beta thalassemia major. Screening of such high-risk groups can considerably reduce the incidence and prevalence of B19 infection; thus, screening is required for epidemiologic surveillance and disease-prevention measures.
Adult ; beta-Thalassemia* ; Blood Transfusion ; Cross-Sectional Studies ; DNA ; Epidemiological Monitoring ; Erythrocytes ; Erythroid Precursor Cells ; Genotype ; Humans* ; Incidence ; Iran* ; Mass Screening ; Parvovirus ; Parvovirus B19, Human* ; Plasma ; Prevalence ; Real-Time Polymerase Chain Reaction ; Thalassemia ; Tropism

OBJECTIVETo construct the ovexpression lentivirus vector of PPP2Cβ, the catalytic subunit of protein phosphatase 2A, so as to obtain high-titer packaged lentivirus particles, and to examine the effect of PPP2Cβ on the erythroid differentiation Methods: The CDS of PPP2Cβ was cloned into the second generation of lentivirus vector FUGW, which should be used to co-transfect HEK 293T cells with the lentiviral expression vector and packaging vectors including pMD2G and pSPAX2. Lentiviruses were harvested at 36 and 48 hours after transfection. Titers of viral stock were determined by using flow cytometric analysis. The Western blot was performed to detect the expression level of PPP2Cβ in K562 cells transinfected with the lentiviruses. Benzidine staining and real-time PCR analysis were used to assess the erythroid differentiation of K562 cells.

RESULTSThe PPP2Cβ overexpressing lentivirus vectors were constructed, the high-titer lentiviral particles were obtained, and then the PPP2Cβ overexpression K562 cell line was established and promote erythroid differentiation of K562 cells.

CONCLUSIONThis study suggests that overexpression PPP2Cβ can promote K562 cell erythroid differentiation.

Cell Differentiation ; Erythroid Cells ; Genetic Vectors ; Humans ; K562 Cells ; Lentivirus ; Protein Phosphatase 2 ; Real-Time Polymerase Chain Reaction ; Transfection

OBJECTIVETo explore the differentiation-inducing potentiality of Pulsatilla saponin A on K562 cells.

METHODSPulsatilla saponin A of different concentrations was used to treat K562 cells; the benzidine staining and the hemoglobinometry were applied to measure the change of hemoglobin content; the flow cytometry (FCM) was used to detect the expression of CD71 and GPA on K562 cells.

RESULTSK562 cells treated with 4 µg/ml pulsatilla saponin A differentiated into the erythroid lineage. With the treatment of pulsatilla saponin A, the hemoglobin content in K562 cells increased significantly; CD71 and GPA expression on the K562 cell surface were up-regulated.

CONCLUSIONPulsatilla saponin A can induce K562 cells to differentiate into erythroid lineage.

Antineoplastic Agents ; Cell Differentiation ; Cell Lineage ; Erythroid Cells ; Humans ; K562 Cells ; Saponins

OBJECTIVETo investigate the effects of oxygen concentration and reactive oxygen species (ROS) on the biological characteristics of hematopoietic stem cells (HSC) and to analyzed the relationship among the oxygen concentration, ROS and the biological characteristics of mouse HSC through simulation of oxygen environment experienced by PB HSC during transplantation.

METHODSThe detection of reactive oxygen species (ROS), in vitro amplification, directional differentiation (BFU-E, CFU-GM, CFU-Mix), homing of adhesion molecules (CXCR4, CD44, VLA4, VLA5, P-selectin), migration rate, CFU-S of NOD/SCID mice irradiated with sublethal dose were performed to study the effect of oxgen concentration and reactive oxygen species on the biological characteristics of mouse BM-HSC and the relationship among them.

RESULTSThe oxygen concentrations lower than normal oxygen concentration (especially hypoxic oxygen environment) could reduce ROS level and amplify more Lin(-) c-kit(+) Sca-1(+) BM HSC, which was more helpful to the growth of various colonies (BFU-E, CFU-GM, CFU-Mix) and to maintain the migratory ability of HSC, thus promoting CFU-S growth significantly after the transplantation of HSC in NOD/SCID mice irradiated by a sublethal dose. BM HSC exposed to oxygen environments of normal, inconstant oxygen level and strenuously thanging of oxygen concentration could result in higher level of ROS, at the same time, the above-mentioned features and functional indicators were relatively lower.

CONCLUSIONThe ROS levels of BM HSC in PB HSCT are closely related to the concentrations and stability of oxygen surrounding the cells. High oxygen concentration results in an high level of ROS, which is not helpful to maintain the biological characteristics of BM HSC. Before transplantation and in vitro amplification, the application of antioxidancs and constant oxygen level environments may be beneficial for transplantation of BMMSC.

Animals ; Cell Differentiation ; Culture Media ; chemistry ; Erythroid Precursor Cells ; cytology ; Granulocyte-Macrophage Progenitor Cells ; cytology ; Hematopoietic Stem Cells ; cytology ; metabolism ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Oxygen ; chemistry ; Reactive Oxygen Species ; metabolism

OBJECTIVETo explore the value of morphological examination, cytochemical staining combined with bone marrow biopsy in the differential diagnosis between myelodysplastic syndrome (MDS) with low blasts and hemolytic anemia (HA).

METHODSThe clinical data of 85 cases of myelodysplastic syndrome with low blasts (< 5%) and 61 patients with hemolytic anemia in Chinese PLA's Gerneral hospital from September 2009 to March 2015 were retrospectively analysed. The clinical characteristics, cytogenetic and molecular features, bone marrow cell count and morphology features, cytochemical staining results and bone marrow biopsy features of above-methioned patients were compared.

RESULTSThere was no significant difference (P > 0.05) in clinical data between MDS group and HA group. Megakaryocytic dysplasia-positive rate, and ring sideroblasts positive rate, and PAS positive rate were significantly higher in MDS group than those that in HA group (P < 0.05). Abnormal localization of immature precursors (ALIP) and megakaryocytic dysplasia positive rate in bone marrow biopsy were significantly higher in MDS group than those that in HA group (P < 0.05), 90.6% of MDS with low blasts patients were identifiable by combined detections.

CONCLUSIONCombining detection of morphology, cytochemistry staining and bone marrow biopsy has been confirmed to be more useful for differential diagnosis between MDS with low blasts and HA.

Anemia, Hemolytic ; complications ; diagnosis ; Biopsy ; Bone Marrow Cells ; cytology ; Diagnosis, Differential ; Erythroid Precursor Cells ; cytology ; Humans ; Megakaryocytes ; cytology ; Myelodysplastic Syndromes ; complications ; diagnosis ; Retrospective Studies ; Staining and Labeling