OBJECTIVETo investigate the roles of telomere and telomerase in the effect of ginsenoside Rg1 to delay hematopoietic stem cell senescence.

METHODSca-1(+) HSC was isolated by magnetic cell sorting(MACS) and divided into five groups: the control group, the aged model group, the Rg1 group, the Rg1 treated aged group and the Rg1 delayed aged group. The changes of cells were observed by senescence-associated beta-Galactosidase (SA-beta-Gal) staining. Cell cycle assay and culture of mixed hematopoietic progenitor cell were used to investigate the effect of ginsenoside Rg1 to delay Sca-1(+) HSC senescence. Telomere length and telomerase activity were detected by southern blotting and TRAP-PCR-SYBR Green staining.

RESULTCompared with aged model group, the percentage of positive cells expressed SA-beta-Gal and the number of cells entered G1 phase were decreased and the number of colony of mixed hematopoietic progenitor was increased. It showed markedly decreased in the shortening of telomere length and reinforcing in the telomerase activity to Rg1 treated aged group and Rg1 delayed aged group. The change of Rg1 delayed aged group was significantly higher than Rg1 treated aged group.

CONCLUSIONActivation of telomerase and prolonging of telomere length might be involved in the process of ginsenoside Rg1 to delay and treat the senescence of Sca-1(+) HSC.

Cellular Senescence ; drug effects ; Ginsenosides ; pharmacology ; Hematopoietic Stem Cells ; drug effects ; physiology ; Telomerase ; metabolism ; Telomere ; drug effects

OBJECTIVETo investigate the anti-aging effect of ginsenoside R1 in serial transplantation of hematopoietic stem cells and progenitor cells.

METHODHSC/HPC aging model in vivo was established through the Sca-1 (+) HSC/HPC serial transplantation of male donor mice that had been separated and purified by the magnetic-activated cell sorting method. The female recipient mice that had been radiated with lethal dose of 60Co gamma ray were divided into four groups: the control group, the aging group, the Rg1-treated aging group and the Rg1 anti-aging group. The expression of Sry genes in bone marrow cells of recipient mice was analyzed by fluorescence quantitative PCR, in order to determine the source of hematopoietic reconstruction cells, observe the survival time and the recovery of the hematology of peripheral blood, and study the reconstruction of the hematopoietic function of recipient mice, the hematopoietic recovery promoted by Rg1, the culture of CFU-Mix of hemopoietic progenitor cells, the cell cycle analysis and aging-related SA-beta-Gal staining analysis on biological characteristics of Sca-1 (+) HSC/HPC aging, and the effect of Rg1 in vivo regulation on Sca-1 + HSC/HPC aging.

RESULTThe hematopoietic reconstruction cells of female recipient mice were derived from male donor mice. With the serial transplantation, the 30-day survival rate and the hematology in peripheral blood of recipient mice decreased. Sca-1 (+) HSC/HPC showed aging characteristics: the ratio of cells in G0/G1 phase and the positive rate of SA-beta-gal staining increased, whereas the number of CFU-Mix decreased. Compared with the aging group of the same generation, Rg1 -treated aging group and Rg1 anti-aging group showed higher 30-day survival rate and WBC, HCT, PLT and CFU-Mix, and lower cell ratio in Sca-1 (+) HSC/HPC G0/G1 stage and positive rate of SA-beta-gal staining. The Rg1 anti-aging group showed more significant changes than the Rg1 -treated aging group.

CONCLUSIONGinsenoside Rg1 has the effect of delaying and treating Sca-1 (+) HSC/HPC aging during the serial transplantation. Rg1 's anti-aging effect is superior to its effect of treating aging.

Aging ; drug effects ; metabolism ; Animals ; Antigens, Ly ; genetics ; metabolism ; Cell Cycle ; drug effects ; Female ; Ginsenosides ; pharmacology ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; cytology ; drug effects ; metabolism ; Humans ; Male ; Membrane Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred C57BL

UNLABELLEDIn this study, the roles of hematopoietic inhibitors elaborated by bone marrow endothelial cells in the proliferation and differentiation of hematopoietic progenitors were investigated. Murine bone marrow endothelial cell conditioned medium (BMEC-CM) was collected and the components with > 10 kD and < 10 kD were obtained by centrifugal ultrafiltration. The effect of BMEC-CM and its components on proliferation of hematopoietic progenitors was evaluated by CFU-GM and HPP-CFC assay and antibody neutralization test. The expression of the inhibitors in BMEC and BMEC-CM was detected by RT-PCR and Western blot, and change of proliferation and differentiation-related genes during expansion of hematopoietic progenitors was examined by membrane hybridization technique.

THE RESULTS(1) When BME C-CM and its components directly were added to CFU-GM and HPP-CFC culture system, BMEC-CM had no effect on colony formation, > 10 kD component enhanced and < 10 kD component inhibited the formation of CFU-GM and HPP-CFC. (2) When BMEC-C M and its components were added to liquid culture system of marrow cells, after 24 hours incubation, CFU-GM decreased and HPP-CFC increased significantly in B MEC-CM group, CFU-GM increased and HPP-CFC had no significant change in > 10 kD component group; and both CFU-GM and HPP-CFC reduced in < 10 kD group. (3) MIP-2, MIP-1 alpha, MSP, TGF-beta, TNF-alpha, IFN-gamma and T beta 4 were expressed in murine marrow endothelial cells, and MIP-2, MIP-1 alpha, MSP, TGF-beta, TNF-alpha and T beta 4 were existed in BMEC-CM. (4) Antibody neutralization test results demonstrated that TGF-beta, MSP, MIP-1 alpha, IFN-gamma and T beta 4 existed in BMEC-CM had significant suppressive effects on CFU-GM and HPP-CFC. (5) T beta 4 combined with 5 hematopoietic cytokines (SCF, IL-3, IL-6, GM-CSF and EPO) added to CD34(+) cells expansion culture system, HPP-CFC significantly increased compared with 5 cytokines group. T beta 4 could downregulated the expression of proliferation and differentiation-related genes and signal transduction-related genes. It is concluded that BM EC-CM promotes the proliferation of early hematopoietic progenitor cells, and this effect is related with the inhibitors existed in BMEC-CM and it could be executed via influencing cell proliferation and differentiation-related genes and signal-related genes.

Animals ; Bone Marrow Cells ; metabolism ; Cell Division ; drug effects ; Cytokines ; genetics ; pharmacology ; Endothelium ; cytology ; metabolism ; Hematopoietic Stem Cells ; drug effects ; physiology ; Mice ; RNA, Messenger ; analysis ; Thymosin ; pharmacology

This study was to investigate the effects of DAPT (N-[N-(3,5-difluorophenacetyl-L:-alanyl)]-S-phenylglycine-butyl ester) on cell cycle, apoptosis, differentiation and expansion of hematopoietic stem cells (HSC) of mouse and to elucidate the possible mechanisms. The mRNA expressions of cell cycle-related genes p18, p21, p27, CDK1, CDK2, CDK4, CDK6, and apoptosis-related genes Bcl-2, Bcl-xl, mcl-1, Bax, Bim, p53, Puma were measured by real-time PCR. The cell cycle and apoptosis of Lin(-)c-kit(+)Sca-1(+) marked cells and CD34(-)Lin(-)c-kit(+)Sca-1(+) marked cells in bone marrow cells were detected by flow cytometry. The differentiation level of HSC was determined by single cell culture. The expansion of HSC were measured with long-term culture. The results indicated that the mRNA expression of the cell cycle related-genes CDK1, CDK2, CDK4, CDK6, p27 in Lin(-) c-kit(+)Sca-1(+) marked cells increased (P < 0.05), the expression of p18, p21 decreased (P < 0.05), the expression of the apoptosis related-genes Bcl-2, Bcl-xl, Bax, p53, Puma in Lin(-) c-kit(+)Sca-1(+) marked cells increased (P < 0.05), the expression of Bim decreased (P < 0.05), the expression of Mcl-1 had not changed (P > 0.05) after treatment with DAPT 1 µmol/L for 5 d. The changes of cell cycle of Lin(-)c-kit(+)Sca-1(+) marked cells in bone marrow had no statistical significance after treatment with DAPT 1 µmol/L for 5 d, CD34(-)Lin(-)c-kit(+)Sca-1(+) marked cells in bone marrow at G(0) phase decreased and at G(1) phase increased after treatment with DAPT 1 µmol/L for 5 d (P < 0.05); the apoptotic fractions of Lin(-) c-kit(+) Sca-1(+) marked cells and CD34(-)Lin(-)c-kit(+)Sca-1(+) marked cells in bone marrow increased after treatment with DAPT 1 µmol/L for 5 d (P < 0.05). The changes of colony number, average number of cells in wells and their differentiation had no statistical significance (P > 0.05) after treatment with DAPT 1 µmol/L for 10 d. Expansion of HSC in bone marrow of mouse decreased after treatment with DAPT 1 µmol/L for 3 d. It is concluded that DAPT not only enhances the exhaustion of CD34(-)Lin(-)c-kit(+)Sca-1(+) marked cells in bone marrow cells of mouse, but also enhances the apoptosis of Lin(-)c-kit(+)Sca-1(+) marked cells and CD34(-)Lin(-)c-kit(+)Sca-1(+) marked cells in bone marrow cells of mouse. DAPT also reduces the expansion of HSC. However, the changes of survival and differentiation of single CD34(-)Lin(-)c-kit(+)Sca-1(+) marked cells in mouse bone marrow cells have no statistical significance.
Animals ; Apoptosis Regulatory Proteins ; metabolism ; Bone Marrow Cells ; metabolism ; Cell Cycle Proteins ; metabolism ; Dipeptides ; pharmacology ; Hematopoietic Stem Cells ; drug effects ; metabolism ; Mice ; Mice, Inbred C57BL ; Signal Transduction

The aim of this study was to investigate the effect of erlotinib on proliferation and differentiation of JAK2V617F-positive cells in vitro, and to provide experimental evidence of erlotinib for potential target therapy in polycythemia vera. Colony forming assays were used to detect the effect of erlotinib on differentiation of hematopoietic progenitor cells from bone marrow of polycythemia vera patients, and MTT method was used to measure the proliferation of HEL cell line containing the JAK2V617F mutation. The results showed that erlotinib 5 µmol/L inhibited the differentiation of JAK2V617F-positive hematopoietic progenitor cells into hematopoietic colonies in vitro, while it had almost no effect on normal hematopoietic progenitor cells from the patients. Erlotinib had inhibitory effect on the proliferation of HEL cell line in a dose dependent manner. The IC(50) was 4.1 µmol/L. It is concluded that erlotinib can inhibit proliferation and differentiation of JAK2V617F-positive cells to a certain extent in vitro.
Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Erlotinib Hydrochloride ; Hematopoietic Stem Cells ; cytology ; Humans ; Janus Kinase 2 ; metabolism ; Polycythemia Vera ; pathology ; Quinazolines ; pharmacology

OBJECTIVETo explore the effects of anti-CD44 monoclonal antibody-IM7 on the in vitro adhesion and migration of chronic myeloid leukemia stem cell (CML-LSC) and its mechanism.

METHODSCD34(+)CD38(-)CD123(+) leukemic stem cells (LSC) from 20 newly-diagnosed chronic myeloid leukemia (CML) patients BM cells and CD34(+)CD38(-) hematopoietic stem cells (HSC) from 20 full-term newborn cord blood cells were isolated with EasySep(TM) magnet beads. The CD44 expression of the LSC and HSC was detected by flow cytometry (FCM), and the adhesion and migration ability of the LSC and HSC pre- and post-incubated with IM7 in vitro by MTT assay and transendothelial migration assay, respectively.

RESULTS(1) After incubated with IM7, the LSC and HSC CD44 expression rates were (86.60 ± 2.10)% vs. (25.40 ± 1.70)% (P < 0.05), respectively. (2) The adhesive ability of the LSC to endothelial cells was decreased markedly after incubated with IM7, the OD value (A(570)) changing from pre-incubation of (0.62 ± 0.11) to post-incubation of (0.34 ± 0.07), while there was little change of A(570) in the HSC group. (3) The migration ability of the LSC group was inhibited evidently after incubated with IM7, the inhibition rate being 46% ∼ 63%, while little change of that in HSC group was detected. (4) The adhesive ability of the LSC group to marrow stromal cells was decreased markedly after incubated with IM7, while little change was found in that of HSC group.

CONCLUSIONThe anti-CD44 monoclonal antibody-IM7 can effectively inhibit the adhesion and migration abilities of the LSC in vitro, which might provide a theoretical evidence for targeting therapy.

Antibodies, Monoclonal ; pharmacology ; Antigens, CD34 ; metabolism ; Bone Marrow ; drug effects ; Flow Cytometry ; Hematopoietic Stem Cells ; drug effects ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; metabolism

OBJECTIVECytomegalovirus (CMV) infection was greatly common in the world. CMV infection produces usually mild or asymptomatic infections in individuals with normal immune responses, whereas it may cause serious disease in immunosuppressive patients. Clinical manifestations include suppression of myelopoiesis, a mononucleosis like syndrome, hepatosplenomegaly, lymphadenopathy, thrombocytopenia, and hemolytic anemia. In patients undergoing bone marrow transplantation CMV remains the most common infectious causes of morbidity and mortality. But the treatment drugs with specific effect for CMV was fewer at the present. This study was to investigate the effect of CMV on proliferation of colony forming unit granulocyte-macrophage (CFU-GM), CFU-erythroid (CFU-E), brust forming unit-erythroid (BFU-E), CFU-multipotential (CFU-Mix) and CFU-megakaryocyte (CFU-Mk) progenitor cells of cord blood (CB) with the presence of ganciclovir (GCV) and astragalus membranaceus in vitro.

METHODSTwenty CB samples were collected from fetal umbilical vein of normal term spontaneous delivery neonates. The colony forming unit-assay was applied to observe the suppression effect of CMV-AD169 strain on CFU-GM, CFU-E, BFU-E, CFU-Mix and CFU-Mk of CB with the presence of GCV and astragalus membranaceus in vitro. The technique of PCR was used to demonstrate the existence of CMV-AD169 DNA in the colony cells of cultured CFU-GM, CFU-E, BFU-E, CFU-Mix and CFU-Mk.

RESULTS(1) The numbers of CFU-GM, CFU-E, BFU-E, CFU-Mix and CFU-Mk colonies in CMV infection groups were significantly less than those in blank and mock group, respectively. The last time of colonies in groups with CMV infection was significantly shorten compared with the blank and mock group. (2) CMV-DNA was positively detected in the colony cells of CMV infection groups by PCR, while negative in the control groups. (3) The lasting time of CFU-GM, CFU-E, BFU-E, CFU-Mix and CFU-Mk colonies infected with CMV extended significantly with the presence of astragalus membranaceus and GCV, and the numbers of those increased significantly compared with the CMV infection group, respectively. The increasing rate of colonies was 27.2%, 45.2%, 49.1%, 39.0% and 11.9% with astragalus membranaceus group, 37.4%, 74.2%, 71.7%, 67.4% and 38.9% with GCV group, 53.6%, 83.8%, 88.7%, 87.8% and 61.5% with astragalus membranaceus and GCV group, respectively.

CONCLUSIONSThe differentiation and proliferation of CFU-GM, CFU-E, BFU-E, CFU-Mix and CFU-Mk were significantly inhibited after infected with CMV-AD169 strain. The suppression effect of CMV-AD169 on CFU-GM, CFU-E, BFU-E, CFU-Mix and CFU-Mk was inhibited with the presence of GCV and astragalus membranaceus in vitro. This suggested that CMV-AD169 may be inhibited or killed by GCV and Astragalus Membranaceus in vitro.

Antiviral Agents ; pharmacology ; Astragalus membranaceus ; chemistry ; Cell Division ; drug effects ; Cytomegalovirus ; drug effects ; Cytomegalovirus Infections ; drug therapy ; Drugs, Chinese Herbal ; pharmacology ; Erythroid Precursor Cells ; cytology ; drug effects ; metabolism ; Fetal Blood ; cytology ; drug effects ; metabolism ; Ganciclovir ; pharmacology ; Hematopoietic Stem Cells ; cytology ; drug effects ; metabolism ; Humans ; Multipotent Stem Cells ; cytology ; drug effects ; metabolism

OBJECTIVETo explore the effect of ligustrazine on the migration of bone marrow mesenchymal stem cells (BMSCs) and protein expressions of matrix metalloproteinase-2 and-9 (MMP-2 and MMP-9) in vitro.

METHODSBMSCs were in vitro isolated and cultured using whole bone marrow adherent method, and phenotypes [surface positive antigens (CD29 and CD90) and negative antigens (CD34 and CD45)] identified using flow cytometry. BMSCs were divided into the blank control group, 25, 50, 100 µmol/L ligustrazine group, and the GM6001 group (100 µmol/L ligustrazine +MMPs inhibitor GM6001 ). The migration of BMSCs was tested by Transwell chamber test and wound healing assay after treated with ligustrazine for 24 h. The protein expressions of MMP-2 and MMP-9 were detected by Western blot.

RESULTSThe third passage BMSCs grew well in uniform morphology. The expression rate of CD29, CD90, CD34, and CD45 was 96.9%, 97.3%, 0.2%, and 3.0%, respectively. Compared with the blank control group, the number of migrated cells and relative distance of cell invasion increased, and the protein expressions of MMP-2 and MMP-9 were elevated in each ligustrazine group (P < 0.05, P < 0.01). Compared with 100 µmol/L ligustrazine group, the number of migrated cells and relative distance of cell invasion decreased in 25 and 50 µmol/L ligustrazine groups and the GM6001 group (P < 0.01). Protein expression of MMP-2 decreased in 25 and 50 µmol/L ligustrazine groups (P < 0.01).

CONCLUSIONLigustrazine could promote the migration of BMSCs in vitro, and its mechanism might be related to up-regulating expression levels of MMP-2 and MMP-9 protein.

Cell Movement ; Cells, Cultured ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Pyrazines ; pharmacology ; Up-Regulation

In peripheral blood hematopoietic stem cell transplantation (PBHSCT) , the mobilization and circulating of bone marrow hematopoietic stem cells in blood with higher oxygen concentration all increase reactive oxygen species(ROS) production, which has negative effect on the biological function of BMHSC. In order to investigate the protective effect of antioxidant on hematopoietic stem cells (HSC), the ascorbic acid 2-phosphate (AA2P), an ascorbic acid derivative of vitamin C, was added in HSC culturing by imitating oxygen conditions which BMHSC experienced in peripheral blood stem cell transplantation. The protective effect of above-mentioned culture methods on the biologic functions of BMHSC was evaluated by vitro amplification assay, committed division assay, reactive oxygen species (ROS) measurement, CD34(+) HSC engraftment. The results showed that the ROS level in HSC from in vitro cultures was much higher than that freshly separated BMHSC, and the amplified AC133(+)CD34(+) HSC, BFU-E, CFU-GM, CFU-GEMM colonies, migration rate and severe combined immunodeficiency (SCID)-repopulating cells (SRC) were all much more than HSC cultured without AA2P. It is concluded that antioxidant intervention may be an effective methods for protecting the biological function of PBHSC and improving the therapeutic effect of PBHSCT.
Antigens, CD34 ; metabolism ; Antioxidants ; pharmacology ; Ascorbic Acid ; analogs & derivatives ; pharmacology ; Cells, Cultured ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Reactive Oxygen Species ; metabolism

OBJECTIVETo determine the role of sphingosine 1-phosphate receptor (S1PRs ) signaling in CD34+ hematopoietic stem/progenitor cell transmigration.

METHODSCD34(+) cells were separated by Ficoll density gradient centrifugation and incubated in DMEM medium with 10% fetal calf serum. The cells were pretreated by FTY720, with or without pertussis toxin (PTX) and antiCXCR4 mAb in the medium, followed by addition of 100 ng/ml SDF-1 into the lower chamber of a Costar 24-well transwell. The migrated cells were counted using FACS and the migrating rates were determined. The expressions of sphingosine 1-phosphate receptors were analyzed in CD34(+) cells before and after the transmigration by reverse transcriptase- polymerase chain reaction (RT-PCR). Cord blood CD34(+) cells were treated with or without FTY720 (10(+) mol/L), and the expressions of CD49d (VLA-4), CD11a (LFA-1), and CD62L (L-selectin) were analyzed at 1, 8, and 16 h after the treatment.

RESULTSWhile FTY720 did not affect spontaneous migration, a substantial increase of SDF-1-induced transmigration was observed in the presence of FTY720 (15.26 2.14 to 28.64 2.37). The FTY720-enhanced transmigration was completely blocked by addition of PTX or antiCXCR4 mAb. S1p1-5 was expressed in fresh isolated cord blood CD34(+) cells. The migrating cells stimulated by FTY720 and SDF-1 only expressed S1P1, S1P3, and S1P4. The expressions of CD49d, CD11a and CD62L on CD34(+) cells treated with FTY720 remained unchanged at the selected time points as compared with the control.

CONCLUSIONSS1PRs are involved the transmigration of CD34(+) cells. The activation of S1PRs results in increased chemotactic response of CD34(+) to SDF-1. These effects are mediated through CXCR4 and PTX-sensitive Gi proteins. Only the CD34(+) cells expressing the specific receptors can rapidly transmigrate. The activation of the S1PRs does not affect the expressions of the adhesion molecules on cord blood CD34(+) cells.

Antigens, CD34 ; metabolism ; Cell Movement ; Cells, Cultured ; Chemokine CXCL12 ; pharmacology ; Fetal Blood ; cytology ; Fingolimod Hydrochloride ; Hematopoietic Stem Cell Mobilization ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Propylene Glycols ; pharmacology ; Receptors, Lysosphingolipid ; metabolism ; physiology ; Signal Transduction ; Sphingosine ; analogs & derivatives ; pharmacology