RESUMEN
Danggui Sini decoction (DSD), a famous Chinese medicine, has been used therapeutically in various diseases. In this study, we tried to investigate whether and how DSD could ameliorate myelosuppression in an animal model, in which myelosuppression is induced by cyclophosphamide treatment. The myelosuppression model was established by intraperitoneal injection of 100 mg/kg cyclophosphamide in mice. Flow cytometry was used to assess cell numbers and evaluate the bone marrow cell cycle distribution. Spleen samples were collected, and the mRNA expression levels of thrombopoietin (TPO) and c-Mpl were analyzed by RT-PCR. Our results demonstrated that DSD could significantly elevate the level of bone marrow hematopoietic stem progenitor cells in myelosuppression mice model. DSD also accelerated cell proliferation by switching cell cycles from G0/G1 phase to S and G2/M phase. Moreover, DSD significantly elevated the mRNA expression level of TPO, but not c-Mpl in spleen. Overall, the present results indicated that DSD is a promising Chinese medicine that is highly potent to ameliorate myelosuppression induced by chemotherapy by upregulating TPO expression.
Asunto(s)
Medicamentos Herbarios Chinos/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Trombopoyetina/metabolismo , Regulación hacia Arriba , Animales , Ciclo Celular , Proliferación Celular , Ciclofosfamida/toxicidad , Células Madre Hematopoyéticas/metabolismo , Masculino , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Bazo/efectos de los fármacos , Bazo/metabolismo , Trombopoyetina/genéticaRESUMEN
OBJECTIVES: Increasing numbers of reports have described hematopoietic improvement after iron chelation therapy in iron-overloaded patients. These observations indicate that excess iron could affect hematopoiesis unfavorably. To investigate how excess iron affects hematopoiesis in vivo, we generated iron-overloaded mice and examined hematopoietic parameters in these mice. METHODS: We generated iron-overloaded mice by injecting 200 mg of iron dextran into C57BL/6J mice, and immature hematopoietic cells in the bone marrow were evaluated by flow cytometric analyses, colony-forming assays, and bone marrow transplantation analyses. We also examined changes in molecular profiles of the hematopoietic microenvironment. RESULTS AND CONCLUSIONS: Iron-overloaded (IO) mice did not show significant defects in the hematopoietic data of the peripheral blood. Myeloid progenitor cells in the bone marrow were increased in IO mice, but the number and function of the erythroid progenitors and hematopoietic stem cells were not significantly affected. However, bone marrow transplantation from normal donors to IO recipients showed delayed hematopoietic reconstitution, which indicates that excess iron impacts the hematopoietic microenvironment negatively. Microarray and quantitative RT-PCR analyses on the bone marrow stromal cells demonstrated remarkably reduced expression of CXCL12, VCAM-1, Kit-ligand, and IGF-1 in the iron-overloaded mice. In addition, erythropoietin and thrombopoietin levels were significantly suppressed, and increased oxidative stress was observed in the IO bone marrow and liver. Consequently, our findings indicate that excess iron can damage bone marrow stromal cells and other vital organs, disrupting hematopoiesis presumably by increased oxidative stress.
Asunto(s)
Células de la Médula Ósea/metabolismo , Médula Ósea/metabolismo , Microambiente Celular/genética , Sobrecarga de Hierro/genética , Hígado/metabolismo , Células Madre Mesenquimatosas/metabolismo , Animales , Médula Ósea/patología , Células de la Médula Ósea/patología , Trasplante de Médula Ósea , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Eritropoyetina/genética , Eritropoyetina/metabolismo , Expresión Génica , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Sobrecarga de Hierro/inducido químicamente , Sobrecarga de Hierro/metabolismo , Sobrecarga de Hierro/patología , Complejo Hierro-Dextran , Hígado/patología , Células Madre Mesenquimatosas/patología , Ratones , Ratones Endogámicos C57BL , Análisis por Micromatrices , Estrés Oxidativo , Factor de Células Madre/genética , Factor de Células Madre/metabolismo , Trombopoyetina/genética , Trombopoyetina/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
OBJECTIVE: Stromal-derived factor-1 (SDF-1/CXCL12) is chemotactic for lympho/hematopoietic stem cells. We have previously shown that increasing peripheral blood (PB) levels of SDF-1 with adenovectors expressing human SDF-1 complementary DNA (ad-SDF-1) leads to hematopoietic stem cell mobilization as well as migration of megakaryocytes and thrombocytosis in mice. Herein, we studied the in vivo effects of ad-SDF-1 and of an analogue peptide of SDF-1 (CTCE-0214) on human hematopoiesis in a xenotransplant model. MATERIALS AND METHODS: Sublethally irradiated (300 cGY) NOD/SCID mice transplanted with human cord blood mononuclear cells (CB MNC) were injected with ad-SDF-1 (10(9) plaque forming units, i.v., x 1) or CTCE-0214 (10 mg/kg/dose, i.v. q 24 hours x 7). Effects on megakaryocytopoiesis (CD41+ cells and platelets) as well as stem cell mobilization were monitored. RESULTS: CB MNC in NOD/SCID mice are able to differentiate into CD41+ cells and platelets, peaking at week 9 at a mean of 3.7 x 10(3)/microL. i.v. injection of ad-SDF-1 increased human CD41+ cells by day 4 in PB and was followed by an increase in human platelet production by day 5, with return to baseline by day 30. Human colony-forming cells (CFC) were mobilized from bone marrow to spleen (by day 6-13) and to PB (by day 13). Human CD34+ and CD33+ cells were mobilized by this treatment as well. A novel SDF-1 peptide agonist (CTCE-0214) also mobilized human CFC and enhanced human thrombopoiesis. CONCLUSION: SDF-1 and its analogue may be of clinical value in stimulating platelet recovery after chemo/radiation treatment as well as in stem cell mobilization.
Asunto(s)
Plaquetas/citología , Quimiocinas CXC/farmacología , Hematopoyesis/efectos de los fármacos , Movilización de Célula Madre Hematopoyética/métodos , Megacariocitos/efectos de los fármacos , Adenoviridae/genética , Animales , Diferenciación Celular/efectos de los fármacos , Quimiocina CXCL12 , Quimiocinas CXC/agonistas , Quimiocinas CXC/sangre , Quimiocinas CXC/genética , Evaluación Preclínica de Medicamentos , Sangre Fetal/citología , Vectores Genéticos/genética , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Quimera por Radiación , Proteínas Recombinantes de Fusión/fisiología , Trombopoyetina/genética , Trombopoyetina/farmacología , Transducción Genética , Trasplante HeterólogoRESUMEN
AIM: To examine the efficacy of saenghyuldan and its components, Ginseng Radix, Paeoniae Radix Alba, and Hominis Placenta extracts (SHD, GR, PRA, and HP, respectively) on the hemopoiesis in a myelosuppression model system. METHODS: Susceptibility to cyclophosphamide (CP) and S180 carcinoma was determined in SHD, GR, PRA, and HP-treated mice. Analysis of peripheral blood and bone marrow cells was demonstrated by changes in cell types and histopathologic examination. The expression of cytokine mRNAs involved in hemopoiesis was examined by RT-PCR. RESULTS: SHD and its separated components (GR, HP, and PRA, respectively) significantly increased the survival in CP- and S180-treated mice. The hematology data demonstrated that all the agents augmented monocyte and leucocyte counts in the peripheral blood and increased bone marrow density and the ratio of leukocyte to erythrocyte in the bone marrow. These findings were positively correlated with the up-regulation of cytokine mRNA expression such as granulocyte colony-stimulating factor (GM-CSF), erythropoietin (EPO), thrombopoietin (TPO), stem cell factor (SCF), and c-Kit. CONCLUSION: SHD is an effective remedy for the bone marrow failure and myelosuppression occurring during chemotherapy.
Asunto(s)
Hematopoyesis/efectos de los fármacos , Materia Medica/farmacología , Paeonia , Panax , Extractos Vegetales/farmacología , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Citocinas/genética , Citocinas/metabolismo , Combinación de Medicamentos , Eritropoyetina/biosíntesis , Eritropoyetina/genética , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Materia Medica/aislamiento & purificación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos ICR , Trasplante de Neoplasias , Paeonia/química , Panax/química , Extractos Vegetales/aislamiento & purificación , Plantas Medicinales/química , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Sarcoma 180/tratamiento farmacológico , Sarcoma 180/patología , Factor de Células Madre/biosíntesis , Factor de Células Madre/genética , Trombopoyetina/biosíntesis , Trombopoyetina/genéticaRESUMEN
OBJECTIVE: Because of the relative inaccessibility of the heart for repeated gene therapy, it would be useful to regulate the expression of transgenes delivered in a single dose of a gene therapy vector. Incorporation into the vector of a regulatable promoter that is responsive to pharmacologic agents that are widely used and well tolerated in clinical practice represents such a control strategy. METHODS: A replication-deficient adenovirus or an adeno-associated virus containing a chimeric promoter composed of 5 glucocorticoid response elements and the murine thrombopoietin complementary DNA (AdGRE.mTPO or AAVGRE.mTPO) was administered to the hearts of Sprague-Dawley rats. Platelet levels were evaluated as a reporter of transgene activity with or without dexamethasone. For comparison, rats received a control adenovirus vector, AdCMV.mTPO or AdCMV.Null, and the control adeno-associated virus vector AAVCMV.luc, which encodes for the firefly luciferase (luc) gene. RESULTS: Platelet elevation in the AdGRE.mTPO group peaked 4 days after dexamethasone administration, with a return to baseline 1 week after the initial corticosteroid dose. Subsequent dexamethasone administration at 2 and 4 weeks resulted in similar but progressively decreased responses. The AAVGRE.mTPO group had 5 peak platelet levels to a minimum of 2.2-fold with respect to baseline without diminution with subsequent dexamethasone administrations out to 169 days. In contrast, the AdCMV.Null and AAVCMV.luc groups demonstrated no increase in platelet counts and the AdCMV.mTPO group demonstrated a slow rise to a single peak platelet count independent of dexamethasone administration. CONCLUSION: It may be possible to control on demand the expression of a gene transferred to the heart. This strategy should be useful in cardiac gene therapy.
Asunto(s)
Antiinflamatorios/administración & dosificación , Dexametasona/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Cardiopatías/terapia , Regiones Promotoras Genéticas/genética , Trombopoyetina/genética , Transgenes/genética , Animales , Antiinflamatorios/farmacocinética , Plaquetas/efectos de los fármacos , Dependovirus/genética , Dexametasona/farmacocinética , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica/genética , Vectores Genéticos/genética , Cardiopatías/genética , Masculino , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/genética , Factores de TiempoRESUMEN
The consequences of long-term in vivo expression of human c-mpl ligand in a mouse model were examined. Transgenic mice expressing the human full-length cDNA in the liver exhibited a fourfold increase in circulating platelet count that persisted stably over the life of the animals. Transgenic animals thrived and appeared healthy for at least 500 days. Transgenic platelets appeared normal with respect to surface antigens and response to platelet aggregation agonists. The highest-expressing transgenic line maintained human c-mpl ligand serum levels of 3 ng/mL. Megakaryocyte numbers in bone marrow and spleen were elevated, as were bone marrow and spleen megakaryocyte colony-forming cells (MEG-CFC). Megakaryocytes were observed in the bone marrow, spleen, liver, and lung, but in no other sites. Circulating myeloid and lymphoid cell populations were increased twofold. Additionally, the animals had a slight but significant anemia despite an increase in marrow colony-forming units-erythroid (CFU-E). No evidence of myelofibrosis was observed in the bone marrow. The platelet nadir in response to administration of either antiplatelet serum (APS) or 5-fluorouracil (5FU) was significantly reduced relative to the control level. Furthermore, the red blood cell (RBC) nadir was reduced relative to control levels in both models, suggesting that c-mpl ligand can directly or indirectly support the maintenance of erythrocyte levels following thrombopoietic insult.