Assuntos
Mieloma Múltiplo , Insuficiência Renal , Humanos , Mieloma Múltiplo/complicações , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/tratamento farmacológico , Bortezomib/efeitos adversos , Talidomida/efeitos adversos , Insuficiência Renal/complicações , Insuficiência Renal/diagnóstico , Dexametasona/efeitos adversosRESUMO
OBJECTIVE: To explore the similarities and variations of biological phenotype and cytotoxicity of human umbilical cord blood natural killer cells (hUC- NK) after human umbilical cord blood-derived mononuclear cells (hUC-MNC) activated and expanded by two in vitro high-efficient strategies. METHODS: Umbilical cord blood mononuclear cells (MNC) from healthy donor were enriched by Ficoll-based density gradient centrifugation. Then, the phenotype, subpopulations, cell viability and cytotoxicity of NK cells derived from Miltenyi medium (denoted as M-NK) and X-VIVO 15 (denoted as X-NK) were compared using a "3IL" strategy. RESULTS: After a 14-day's culture, the contents of CD3-CD56+ NK cells were elevated from 4.25%±0.04% (d 0) to 71%±0.18% (M-NK) and 75.2%±1.1% (X-NK) respectively. Compared with X-NK group, the proportion of CD3+CD4+ T cells and CD3+CD56+ NKT cells in M-NK group decreased significantly. The percentages of CD16+, NKG2D+, NKp44+, CD25+ NK cells in X-NK group was higher than those in the M-NK group, while the total number of expanded NK cells in X-NK group was half of that in M-NK group. There were no significant differences between X-NK and M-NK groups in cell proliferation and cell cycle, except for the lower percentage of Annexin V+ apoptotic cells in M-NK group. Compared with X-NK group, the proportion of CD107a+ NK cells in M-NK group were higher under the same effector-target ratio (Eâ¶T) (P<0.05). CONCLUSION: The two strategies were adequate for high-efficient generation of NK cells with high level of activation in vitro, however, there are differences in biological phenotypes and tumor cytotoxicity.
Assuntos
Sangue Fetal , Células Matadoras Naturais , Humanos , Linfócitos T , Leucócitos Mononucleares/metabolismo , Proliferação de Células , Antígeno CD56/metabolismoRESUMO
OBJECTIVE: To investigate the regulatory effect and mechanism of DNA methyltransferase 3A (DNMT3a) in hydroquinone-induced hematopoietic stem cell toxicity. METHODS: Cells (HSPC-1) were divided into 4 groups, that is A: normal HSPC-1; B: HQ-intervented HSPC-1; C: group B + pcDNA3 empty vector; D: group B + pcDNA3- DNMT3a. RT-qPCR and Western blot were used to detect the expression levels of DNMT3a and PARP-1 mRNA and protein, respectively. Cell morphology was observe; Cell viability and apoptosis rate of HSPC-1 were detected by MTT and flow cytometry, respectively. RESULTS: Compared with group A, the expression levels of DNMT3a mRNA and protein in HSPC-1 of group B were decreased, while PARP-1 mRNA and protein were increased (P<0.05); there was no significant difference in the above indexes between group C and group B; compared with group B, the expression levels of DNMT3a mRNA and protein showed increased, while PARP-1 mRNA and protein were decreased significantly in cells of group D transfected with DNMT3a (P<0.05). Cells in each group were transfected with DNMT3a and cultured for 24 h, HSPC-1 in group A showed high density growth and mononuclear fusion growth, while the number of HSPC-1 in group B and C decreased and grew slowly. Compared with group B and C, the cell growth rate of group D was accelerated. The MTT analysis showed that cell viability of HSPC-1 in group B were lower than that of group A at 24 h, 48 h and 72 h (P<0.05); after transfected with DNMT3a, the cell viability of HSPC-1 in group D were higher than that of group B at 24 h, 48 h and 72 h (P<0.05). The apoptosis rate of cells in group B was significantly higher than that of group A (P<0.001), while the apoptosis rate in group D was lower than that of group B (P<0.001). CONCLUSION: DNMT3a may be involved in the damage of hematopoietic stem cells induced by hydroquinone, which may be related to the regulation of PARP-1 activity by hydroquinone-inhibited DNMT3a.
Assuntos
DNA Metiltransferase 3A , Células-Tronco Hematopoéticas , Hidroquinonas , Apoptose , Proliferação de Células , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Hidroquinonas/toxicidade , Poli(ADP-Ribose) Polimerase-1 , RNA Mensageiro/metabolismoRESUMO
OBJECTIVE: To investigate regulatory T cells (Tregs) relative content in peripheral blood and bone marrow of patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) treated with or without decitabine (DAC), analyze the immunomodulatory of Tregs in pathogenesis and remission of MDS and AML, as well as effect of DAC on Tregs. METHODS: From October 2018 to February 2019, 15 patients with MDS and 49 patients with AML (newly diagnosed, treated with DAC or other chemotherapy regimens) were enrolled in this study, and 14 cases with iron deficiency or megaloblastic anemia while without malignant tumor and autoimmune disease as controls. The Tregs relative contents in bone marrow and peripheral blood were analyzed by flow cytometry, meanwhile clinical data of the objects were collected. RESULTS: In peripheral blood and bone marrow of the patients with MDS and AML, the Tregs relative contents at newly diagnosed were higher than those of the control group (P=0.05, P=0.043). The Tregs relative content of AML patients in DAC regimen treatment group was significantly lower than that in the newly diagnosed group and non-DAC chemotherapy group (P<0.05). In DAC regimen treatment group, the Tregs relative contents was significantly lower in remission group than in non-remission group (P<0.05). There was no difference between DAC regimen treatment group and control group in Tregs relative content. CONCLUSION: DAC may increase the body's anti-tumor immunity by consuming Tregs content, enhance the body's immune function to identify and kill tumor cells, thereby promote the patients' reliefs.
Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Protocolos de Quimioterapia Combinada Antineoplásica , Medula Óssea , Decitabina/uso terapêutico , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Síndromes Mielodisplásicas/tratamento farmacológico , Linfócitos T Reguladores , Resultado do TratamentoRESUMO
OBJECTIVE: To investigate the effects of human amniotic mesenchymal stem cellï¼AMSCï¼ on acute graft-versus-host disease (aGVHD) in xenotransplatation. METHODS: NPG mice were injected with human PBMNC via tail vein to establish a xenografted aGVHD model. The mice in the experimental group were divided into PBMNC infusion group and PBMNC+AMSC co-infusion group, the general condition, survival time and manifestations of aGVHD were observed, the body weight and blood routine indicators were detected, the pathological changes of aGVHD target organs (lung, liver, spleen, small intestine) were observed by HE staining, and the levels of human T cells in peripheral blood, tissues and organs of mice was detected by flow cytometry. RESULTS: The manifestations of aGVHD (lassitude hunchback, shrub, weight reduction, etc.) and the pathological damage of the target organs (lung, liver, spleen, intestine) in PBMNC+AMSC co-infusion group were lighter than those in PBMNC infusion group. Moreover, the PBMNC and AMSC co-infusion significantly reduced the implantion proportion of human T lymphocytes (CD3+, CD45+) in mice and increased the ratio of CD4+/CD8+. CONCLUSION: Infusion of human-derived AMSC can attenuate the manifestations of aGVHD in mouse xenografts to a certain level, and improve the pathological damage of receptor target organs.
Assuntos
Doença Enxerto-Hospedeiro , Células-Tronco Mesenquimais , Doença Aguda , Animais , Xenoenxertos , Humanos , Camundongos , Linfócitos T , Transplante HeterólogoRESUMO
The aim of the present study was to investigate the effect of coculture with amniotic epithelial cells (AECs) on the biological characteristics of amniotic mesenchymal stem cells (AMSCs), to compare the expression of CXC motif chemokine receptor 4 (CXCR4) in cocultured AMSCs and to investigate the roles of the stromal cellderived factor1 (SDF1)/CXCR4 axis in the homing and migration of AMSCs. AMSCs were isolated from human amniotic membranes, purified and then differentiated into osteoblasts and adipocytes in vitro, which was verified by von Kossa Staining and Oil Red O staining. Cell viability was measured by Cell Counting kit8 and trypan blue assays at 24, 48 and 72 h, the expression of CXCR4 was analyzed by immunofluorescencebased flow cytometry and reverse transcriptionquantitative polymerase chain reaction, and the migration ability of AMSCs in vitro was observed by a migration assay. The results demonstrated that cell viability (at 48 and 72 h) and survival (at 24, 48 and 72 h) in the coculture and serum groups were higher compared with the serumfree group. Furthermore, CXCR4 mRNA and protein expression, and migration along the SDF1 gradient, in the coculture and serumfree groups were higher compared with the serum group. Overall, the results indicated that AMSCs cocultured with AECs exhibited enhanced proliferation activity and survival rate. In conclusion, the present study demonstrated that coculture of AMSCs with AECs upregulated CXCR4 on the surface of AMSCs and enhanced the migration ability of AMSCs in vitro. This result may improve the directional migration and homing ability of AMSCs, as well as provide a theoretical basis for the application of AMSCs in clinical practice as a novel strategy to increase the success of hematopoietic stem cell transplantation.
Assuntos
Adipócitos/metabolismo , Âmnio/metabolismo , Diferenciação Celular , Movimento Celular , Células Epiteliais/metabolismo , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/metabolismo , Adipócitos/citologia , Adulto , Âmnio/citologia , Quimiocina CXCL12/metabolismo , Técnicas de Cocultura , Células Epiteliais/citologia , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Receptores CXCR4/metabolismoRESUMO
OBJECTIVE: To explore the relationship between TNF-alpha, transcriptional co-activator with PDZ-binding motif (TAZ) and bone disease of multiple myeloma. METHODS: The biological characteristics, especially the osteogenic potential of marrow MSCs from myeloma patients and normal subjects were studied. Real-time RT-PCR and Western-blot were employed to detect mRNA and protein expression of TAZ in MSCs. The concentration of TNF-alpha in the marrow plasma was detected using ELISA method. CD138(+) myeloma cells were cocultured with normal MSCs with or without anti-human TNF-alpha monoclonal antibody in the Transwell system. Real-time RT-PCR was employed to detect the mRNA expressions of ALP, Cbfa1 and TAZ in MSCs two weeks later. von Kossa staining was used to detect the mineral deposition. TNF-alpha was added into the culture media of normal marrow MSCs and real-time RT-PCR and Western-blot were employed to detect mRNA and protein expression of TAZ in MSCs one week later. RESULTS: Real-time RT-PCR revealed that the mRNA of osteogenic markers was decreased in comparison with that of normal controls after cultured in the osteogenic medium. von Kossa staining showed weakened mineral deposition in MSCs from multiple myeloma patients compared with that in normal subjects after osteogenic differentiation for two weeks. The mRNA and protein levels of TAZ in the MSCs from myeloma patients were decreased. TNF-alpha concentration in the marrow plasma of myeloma patients was higher than that in the normal controls [(355.4 +/- 49.1) vs. (92.3 +/- 17.2) pg/ml]. CD138(+) myeloma cells inhibited mRNA expressions of ALP, Cbfal1 and TAZ in MSCs, which could be partially reversed by anti-human TNF-alpha monoclonal antibody. CONCLUSION: The osteogenic potential of MSCs from myeloma patients is significantly decreased in comparison with that in normal subjects, which may play an important role in the pathology of myeloma bone disease. TAZ expression inhibited by TNF-alpha may play an important role in this inhibition effect.
Assuntos
Células-Tronco Mesenquimais/metabolismo , Mieloma Múltiplo/patologia , Osteogênese , Fatores de Transcrição/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Aciltransferases , Adulto , Idoso , Fosfatase Alcalina/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Pessoa de Meia-Idade , Mieloma Múltiplo/metabolismo , Osteocalcina/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição/genética , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/imunologiaRESUMO
The aim of this study was to explore the changes in cellular senescence related indexes of bone marrow mesenchymal stem cells (BMMSCs) after total body irradiation (TBI). At different time points after 4 Gy irradiation, BMMSCs were isolated from male C57BL/6 mice and cultured. Morphology, senescence-associated beta-galactosidase (SA-beta-gal) staining and cell cycle analysis were used to evaluate the changes in BMMSCs at cellular level while real-time RT-PCR was used to detect the alterations in senescence related gene expression including p16INK4a, p21Cip1/Waf1, p53 and TGF-beta1. The results showed that within 4 weeks after exposure to 4 Gy TBI, the morphology of BMMSCs and the expression level of SA-beta-gal were not significantly changed, the cellular senescence-related cell cycle arrest was not occurred and the senescence related gene expression level was not increased. It is concluded that at the early stage after 4 Gy TBI, the related molecular level of cellular senescence in BMMSCs is not changed.
Assuntos
Células da Medula Óssea/efeitos da radiação , Senescência Celular/efeitos da radiação , Células-Tronco Mesenquimais/efeitos da radiação , Irradiação Corporal Total , Animais , Células da Medula Óssea/citologia , Células Cultivadas , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
To investigate the effect of irradiation on the quantity and osteogenesis potential of BMMSCs and to explore the response of them in the irradiation stress and its contribution to long-term effects of radiation-induced bone and hematologic injury, a total body irradiation (TBI) murine model was adopted. The number of CFU-F and cell cycle profile of BMMSCs were analyzed at different time points before and after TBI. Osteogenic differentiation was evaluated by Von Kossa staining, expressions of osteogenesis-related genes and transcriptional coactivator with PDZ-binding motif (TAZ) were detected by real-time RT-PCR. The results showed that the number of CFU-F decreased greatly at day 28 after TBI. At day 3 after TBI, more cells entered cell cycle and the osteogenesis potential was greatly enhanced followed by recovery of cell cycle distribution and significant defect in osteoblast differentiation respectively, meanwhile the expression of TAZ was changed. It is concluded that TBI results in the reduction of bone marrow mesenchymal stem/progenitor cell pool and alters the osteogenesis potential of BMMSCs, which is related to the change of TAZ expression.
Assuntos
Células da Medula Óssea/efeitos da radiação , Células-Tronco Mesenquimais/efeitos da radiação , Osteogênese/efeitos da radiação , Irradiação Corporal Total , Animais , Células da Medula Óssea/citologia , Ensaio de Unidades Formadoras de Colônias , Relação Dose-Resposta à Radiação , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
OBJECTIVE: To isolate and culture bone marrow mesenchymal stem cells (MSCs) from chronic myelogenous leukemia (CML) patients and examine their functional characteristics. METHODS: Bone marrow was extracted from the anterior superior iliac spines of 21 patients with CML. MSCs were isolated and cultured. Single colony derived MSCs were harvested by limiting dilution. The cell cycle and immunophenotype of the expanded clonal MSCs were detected by fluorescence-activated cell sorter (FACS). Different agents were used to induce the MSCs to differentiate into osteocyte, adipocyte and neural cells. Von Kossa staining, oil-red staining, and Western blotting was used to examine the ability of differentiation. PCR was used to detect the expression of BCR/ABL gene. The ultrastructure of the CML derived MSCs was observed with electron microscopy. Sixteen BALB/c nude mice were randomly divided into 2 equal groups to be inoculated with HL60 cancer cells and CML derived MSCs to observe the tumorigenicity. MSCs were cultured in soft agar for 2 weeks to observe the clone growth. RESULTS: Fibroblast-like, positive in CD29, CD44, and CD105, and negative in CD116, CD34, CD48, and HLA-DR, the CML derived MSCs could differentiate into osteocyte, adipocyte and neural cells. CML derived MSCs showed normal karyotype and ultrastructure, they did not express BCR/ABL gene. After 2 weeks' culture no clone was formed from the MSCs. Four weeks after tumors were shown in 6 of the 8 mice inoculated with HL60 cells, and no tumor was seen in the mice inoculated with MSCs. CONCLUSION: Able to differentiate into different types of cell and without tumorigenicity, MSCs from the bone marrow of CML patients have the potentiality in clinical application.
Assuntos
Células da Medula Óssea/patologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/patologia , Células-Tronco Mesenquimais/fisiologia , Animais , Células da Medula Óssea/fisiologia , Diferenciação Celular , Separação Celular , Células Cultivadas , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/cirurgia , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Distribuição AleatóriaRESUMO
Fibrosis is the common end stage of most liver diseases. Unfortunately, there is no effective treatment available currently. This study was designed to evaluate the effect of Flk1+ mesenchymal stem cells (MSC) from murine bone marrow (Flk1 + MSC) on fibrosis formation induced by carbon tetrachloride (CCl4). In this study Flk1+ MSC were isolated from bone marrow of male BALB/c mice. A CCl4 induced hepatic fibrosis model was used. Flk1+ MSC were systemically infused immediately or one week after the female mice were challenged with CCl4. Fibrosis index and donor cell engraftment were assessed two or five weeks after CCl4 challenge. We found that Flk1+ MSC transplantation immediately, but not one week after exposure to CCl4, significantly reduced CCl4-induced liver damage and collagen deposition. In addition, levels of hepatic hydroxyproline and serum fibrosis markers (HA, P-III-P) in mice receiving immediate Flk1+ MSC transplantation after CCl4 challenge were significantly lower compared to those of control mice. More importantly, histological examination suggested that hepatic damage recovery was much better in these immediately Flk1+ MSC-treated mice. Immunofluorescence, PCR, and fluorescence in situ hybridization (FISH) analysis revealed that donor cells engrafted into host liver, had epithelium-like morphology and expressed albumin (ALB), although at low frequency. In conclusion Flk1+ MSC might initiate endogenous hepatic tissue regeneration, engraft into host liver in response to CCl4 injury, and ameliorate its fibrogenic effects.