RESUMO
OBJECTIVE: To investigate the effect of human umbilical cord-derived mesenchymal stem cells (UC-MSC) on the differentiation of leukemic cells. METHODS: The co-culture system of UC-MSC with acute promyelocytic leukemic cell line NB4 cells was constructed in vitro,and the differentiation status of the leukemic cells was assessed by cell morphology,nitroblue tetrazolium reduction test,and cell surface differentiation marker CD11b. RESULTS: UC-MSC induced the granulocytic differentiation of NB4 cells. When UC-MSC and a small dose of all-trans retinoic acid were applied together,the differentiation-inducing effect was enhanced in an additive manner. Interleukin (IL)-6Ra neutralization attenuated differentiation and exogenous IL-6-induced differentiation of leukemic cells. CONCLUSION: UC-MSC can promotd granulocytic differentiation of acute promyelocytic leukemia cells by way of IL-6 and presented additive effect when combined with a small dose of all-trans retinoic acid.
Assuntos
Diferenciação Celular , Interleucina-6/metabolismo , Leucemia Promielocítica Aguda/patologia , Células-Tronco Mesenquimais/metabolismo , Linhagem Celular Tumoral , Humanos , Tretinoína/farmacologia , Cordão Umbilical/citologiaRESUMO
OBJECTIVE: To construct a method to culture pancreatic progenitor cells in hanging drop and on floating filter,and to examine if pancreatic progenitor cells can differentiate into mature endocrine cells with this method. METHODS: Murine embryos at day 12.5 were isolated and digested into single cells,which were then cultured in hanging drop for 24h and formed spheres.Spheres were cultured on the filter for 6 days,which floated in the dish containing medium.During culture,the expressions of pancreas duodenum homeobox-1(PDX-1)and neurogenin3(Ngn3)were determined.The expressions of endocrine and exocrine markers,insulin,glucagon,and carboxypeptidase(CPA)were determined on day 7 by immunohistochemistry.Insulin secretion of spheres stimulated by glucose was detected by ELISA.The changes of pancreatic marker expressions during culture were monitored by real-time polymerase chain reaction(PCR). RESULTS: One day after the culture,there were still a large amount of PDX-1 positive cells in pancreatic spheres,and these cells proliferated.On day 3,high expression of Ngn3 was detected,and the Ngn3-positive cells did not proliferate.On day 7,The expressions of endocrine and exocrine markers in the differentiated pancreatic progenitor cells were detected,which were consistent with that in vivo.Insulin was secreted by spheres upon the stimulation of glucose. CONCLUSION: In hanging drop and on floating filter,pancreatic progenitor cells can differentiate into mature endocrine cells.
Assuntos
Técnicas de Cultura de Células , Células Endócrinas/citologia , Pâncreas/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular , Células Cultivadas , Proteínas de Homeodomínio/metabolismo , Insulina/metabolismo , Camundongos , Transativadores/metabolismoRESUMO
OBJECTIVE: To establish a new culture method to induce the differentiation of embryonic pancreatic cells into mature endocrine cells. METHODS: Mouse embryos at day 12.5 were used and embryonic pancreata were isolated. The isolated embryonic pancreata were cultured on the filter for 7 days, which floated in the dish containing medium. During culture, the expression of pancreas duodenum homeobox-1 (PDX-1), a pancreatic stem cell marker, was examined at day 1. The expression of neurogenin 3 (Ngn3), a pancreatic progenitor cell marker, was examined at day 3. The expressions of endocrine and exocrine markers, insulin, glucagon, and carboxypeptidase (CPA) were examined at day 7 by immunohistochemistry. The kinetics of pancreatic marker expression during culture was assayed by real-time PCR. RESULTS: Many pancreatic stem cells still existed in embryonic pancreata cultured for 1 day; meanwhile, these pancreatic stem cells proliferated in high rate. A large amount of pancreatic progenitor cells were found in embryonic pancreata cultured for 3 days.Pancreatic stem/progenitor cells differentiated into mature endocrine and exocrine cells in embryonic pancreata after having been cultured for 7 days. Furthermore, the expression pattern of pancreatic marker is consistent with that in vivo. CONCLUSION: We successfully established a new culture method, with which embryonic pancreatic cells can efficiently differentiate into mature endocrine cell.
Assuntos
Técnicas de Cultura de Células , Diferenciação Celular , Embrião de Mamíferos , Células Endócrinas/citologia , Pâncreas/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Cultivadas , Feminino , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Transativadores/metabolismoRESUMO
PI3K signaling pathway plays a significant role in embryonic stem cells (ES cells) self-renewal. Overexpression of Nanog maintains mouse ES cells pluripotency independent of leukemia inhibitory factor (LIF). However, little is known about the effect of PI3K signaling pathway on ES cells with Nanog overexpression. Our experiments aimed to explore the relationship between PI3K signaling pathway and Nanog expression in ES cells. We observed the effect of LY294002, a specific inhibitor of PI3K pathway, on wild-type J1 cells and Nanog overexpressing (Ex-Nanog) J1 cells in the presence or absence of LIF. With LY294002 treatment, both of them lost their ES features even in the presence of LIF. But the differentiation induced by LY294002 on Ex-Nanog J1 cells was slighter lower than that on wild-type J1 cells. These results indicate that inhibition of PI3K pathway induces mouse ES cells differentiation. Exogenous Nanog sustains mouse ES cells pluripotency independent of LIF, and alleviates the differentiation induced by LY294002. But it is insufficient to totally reverse the differentiation.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/fisiologia , Proteínas de Homeodomínio/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Células-Tronco Pluripotentes/fisiologia , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Cromonas/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Fator Inibidor de Leucemia/farmacologia , Camundongos , Morfolinas/metabolismo , Proteína Homeobox Nanog , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Células-Tronco Pluripotentes/citologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
AIM: The aim of the study was to investigate whether lovastatin restores the survival and function of endothelial progenitor cells (EPCs) damaged by oxLDL. METHODS: EPCs were preincubated with different concentrations of lovastatin (2, 10, and 50 micromol/L) with or without the Akt inhibitor triciribine for 24 h and were then exposed to 50 microg/mL oxLDL for 48 h. The survival of EPCs, as well as the cellular migration, adhesion, and tube formation of these cells, was examined. To explore the mechanisms of lovastatin's effects on EPCs, the levels of phosphorylated Akt and eNOS and of total eNOS protein and mRNA were assayed. RESULTS: Incubation of EPCs with oxLDL resulted in significant apoptosis and impaired cellular migration, adhesion and tube structure formation. The detrimental effects of oxLDL on EPC survival and function were attenuated by pretreatment of EPCs with lovastatin. However, when EPCs were pretreated with lovastatin and triciribine at the same time, the beneficial effects of lovastatin were abolished by triciribine. Furthermore, oxLDL caused a significant downregulation of eNOS mRNA and protein expression, as well as a suppression of Akt and eNOS phosphorylation. However, the effects of oxLDL on Akt/eNOS activity and eNOS expression were reversed by lovastatin. CONCLUSION: Lovastatin reverses the survival and function of EPCs by regulating the Akt/eNOS signaling pathway and the gene transcription of eNOS.
Assuntos
Células-Tronco Hematopoéticas , Lipoproteínas LDL/efeitos adversos , Lovastatina/farmacologia , Antígenos CD34/imunologia , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo , Feminino , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação/efeitos dos fármacos , Gravidez , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The title compound, (C(10)H(10)N(2))(2)[SiW(12)O(40)]·C(10)H(8)N(2)·6H(2)O or (4,4'-bipyH(2))(2)[SiW(12)O(40)].(4,4'-bipy)·6H(2)O (4,4'-bipy is 4,4'-bipyridine), was prepared under hydro-thermal conditions. The asymmetric unit contains a discrete Keggin-type [SiW(12)O(40)](4-) anion (located on a twofold axis), one 4,4'-bipy (located on a twofold axis), two (4,4'-bipyH(2))(2+) cations and six uncoordinated water mol-ecules. The polyoxoanion is constructed from a central SiO(4) tetra-hedron which shares its O atoms with four trinuclear W(3)O(13) groups, each of which is made up of three edge-sharing WO(6) octa-hedra. The water mol-ecules and [SiW(12)O(40)](4-) anions are linked through hydrogen bonds.
RESUMO
The title compound, [Fe(C(12)H(8)N(2))(3)](3)[FeW(12)O(40)]·2H(2)O, was prepared under hydro-thermal conditions. The discrete Keggin-type [FeW(12)O(40)](6-) heteropolyoxoanion has threefold symmetry, with the Fe(II) atom located on the threefold rotation axis. The central FeO(4) tetra-hedron in the anion shares its O atoms with four W(3)O(13) trinuclear units, each of which is made up of three edge-shared WO(6) octa-hedral units. The Fe(II) atom in the complex cation, viz [Fe(phen)(3)](2+) (phen is 1,10-phen-anthroline), shows a slightly distorted octa-hedral geometry defined by six N atoms from three phen ligands. The polyoxoanions pack together with the cations, with the disordered water mol-ecules located in voids; the site occupancy factor for each water O atom is 0.33.
RESUMO
OBJECTIVE: To investigate whether oxidized low-density lipoprotein (oxLDL) affects the survival and activity of endothelial progenitor cell (EPC) and whether the effects are mediated by lectin-like oxidized low-density lipoprotein receptor (LOX-1). METHODS: CD34+ cells isolated from human umbilical blood were cultured in endothelial cell growth medium-2 (EGM-2). After 14 days of culture, some EPCs were stimulated with 10, 25, 50 microg/ml of oxLDL for 48 hours; some were preincubated with LOX-1 mAb, a blocking antibody of LOX-1, for 24 hours, then exposed to 50 microg/ml oxLDL for 48 hours; others without any further treatment were used as control. The survival of EPC and the ability of adhesion, migration, and tube formation were examined. The levels of LOX-1 protein and mRNA expression were also assayed. RESULTS: Incubation with oxLDL at concentrations of 25 microg/ml or higher resulted in a dose-dependent increase of EPC apoptosis [25 microg/ml: (15.8 +/- 1.1.0%, 50 microg/ml: (18.8 +/- 2.0)% versus control: (9.0 +/- 1.2)%; P < 0.05]. Treated with oxLDL led to a significantly reduced migratry rate [25 microg/ml: (5.7 +/- 1.0)%, 50 microg/ml: (5.1 +/- 0.8)% versus control: (9.5 +/- 0.8)%; P < 0.05]. EPC treated with oxLDL showed a dose-dependent reduction of adhesion to fibronectin (25 Kg/ml: 33 +/- 2, 50 microg/ml: 30 +/- 3 versus control: 37 +/- 5; P < 0.05). Treatment with oxLDL impaired the in vitro vasculogenesis ability of EPCs. The total length of the tube structures in each photograph was decreased [25 microg/ml: (2.9 +/- 0.5) mm, 50 microg/ml: (1.8 +/- 0.5) mm versus control: (5.0 +/- 0.6) mm; P < 0.05]. The tube structure was severely disrupted, resulting in an incomplete and sparse tube network. However, all the detrimental effects on EPC were attenuated by pretreatment of EPC with LOX-1 mAb. In addition, Western blot analysis revealed that oxLDL increased LOX-1 protein expression from 100% to (172 +/- 8)% at a dose of 50 microg/ml. Furthermore, oxLDL caused an increase in LOX-1 mRNA expression from 100% to (174 +/- 39)% at a dose of 50 microig/ml. CONCLUSION: OxLDL can directly inhibit EPC survival and activity and these effects are mediated by its receptor, LOX-1.
Assuntos
Células Endoteliais/fisiologia , Lipoproteínas LDL/fisiologia , Receptores Depuradores Classe E/fisiologia , Células-Tronco/fisiologia , Antígenos CD34/metabolismo , Apoptose , Adesão Celular , Movimento Celular , Sobrevivência Celular , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Sangue Fetal/citologia , Humanos , Lipoproteínas LDL/farmacologia , Neovascularização Fisiológica , Receptores Depuradores Classe E/biossíntese , Células-Tronco/efeitos dos fármacosRESUMO
OBJECTIVE: To investigate the association between Akt/eNOS signal pathway changes and the survival/function of endothelial progenitor cells (EPC) in the presence of oxLDL, L-NAME or triciribine. METHODS: After 14 d culture, EPC was stimulated with different concentrations of oxLDL, L-NAME or triciribine for 48 h. In one group, EPC was preincubated with L-arginine for 24 h and then exposed to 50 microg/ml oxLDL for 48 h. The survival and the ability of adhesion, migration and tube structure formation of EPC were observed and the level of phosphorylated Akt protein expression, eNOS protein and mRNA expression were assayed. RESULTS: Incubation with oxLDL at concentration 25 microg/ml or higher resulted EPC apoptosis, significantly reduced migratory rate, reduced adhesion to fibronectin and impaired ability of EPC to form tube structure in a dose-dependent manner. A simultaneous dose-dependent NO generation and Akt phosphorylation decrease as well as eNOS expression reduction at protein and mRNA levels were also observed. Pretreatment of EPC with L-arginine could attenuate these changes. CONCLUSION: oxLDL reduced EPC survival and function via regulating Akt/eNOS signal pathway.
Assuntos
Células Endoteliais/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Células Cultivadas , Células Endoteliais/citologia , Humanos , Lipoproteínas LDL/metabolismo , Fosforilação , Células-Tronco/citologia , Veias Umbilicais/citologiaRESUMO
Mesenchymal stem cells (MSCs) have shown promise for use in cell therapy, and due to their tumor tropism can serve as vehicles for delivering therapeutic agents to tumor sites. Because interleukin-8 (IL-8) is known to mediate the protumor effect of MSCs, elimination of IL-8 secretion by MSCs may enhance their safety for use in cancer gene therapy. However, little is known concerning the effect of endogenously secreted IL-8 on MSCs. We performed studies using placenta-derived MSCs (PMSCs) to determine whether knockdown of IL-8 would influence their biological activity. We first verified that IL-8 and its membrane receptor CXCR2, but not CXCR1, were highly expressed in PMSCs. We then employed lentivirus-mediated small hairpin RNA interference to generate stable IL-8-silenced PMSCs, which displayed a variety of characteristic senescent phenotypes. We observed that at day 9 post-transfection, IL-8-silenced PMSCs had become larger and displayed a more flattened appearance when compared with their controls. Moreover, their proliferation, colony forming unit-fibroblast formation, adipogenic and osteogenic differentiation, and immunosuppressive potentials were significantly impaired. Enhanced senescence-associated ß-galactosidase (SA-ß-gal) activity and specific global gene expression profiles confirmed that IL-8 silencing evoked the senescence process in PMSCs. Increased levels of p-Akt and decreased levels of FOXO3a protein expression suggested that reactive oxygen species played a role in the initiation and maintenance of senescence in IL-8-silenced PMSCs. Notably, the majority of CXCR2 ligands were downregulated in presenescent IL-8-silenced PMSCs but upregulated in senescent cells, indicating an antagonistic pleiotropy of the IL-8/CXCR2 signaling pathway in PMSCs. This effect may promote the proliferation of young cells and accelerate senescence of old cells.
Assuntos
Proliferação de Células/genética , Senescência Celular/genética , Técnicas de Silenciamento de Genes , Interleucina-8/genética , Células-Tronco Mesenquimais/metabolismo , Placenta/metabolismo , Feminino , Humanos , Interleucina-8/metabolismo , Células-Tronco Mesenquimais/citologia , Placenta/citologia , Gravidez , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismoRESUMO
Human umbilical cord mesenchymal stromal cells (hUC-MSCs) are currently being used as novel therapeutic agents in numerous clinical trials. Previous works have shown that hUC-MSCs possess profound immunomodulatory capacities through IL-1 stimulation produced by peripheral blood mononuclear cells (PBMCs), their main cellular partner in most pathophysiological and therapeutic situations. The present study was designed to explore the role of TNF-α in these interactions. In these experiments, we demonstrated that TNF-α originated from PBMCs under the influence of IL-1. We also showed that TNF-α acted differently depending upon the concentrations reached. At low concentrations it clearly contributed to IL-6 and monocyte chemotactic protein 1 (MCP-1) production. At high concentrations, used alone or in association with the TNF-related apoptosis-inducing ligand, TNF-α also stimulated hUC-MSC IL-6 but, more intensely, MCP-1 production. This stimulation was associated but independent of apoptosis induction in a process involving Inhibitor of Apoptosis Proteins. Interferon gamma (IFN-γ), tested to stimulate PBMC and tissue activation, amplified IL-6 and MCP-1 production and cell death by, apparently, a different process involving necrosis. Our findings bring new insights into the complex interactions between hUC-MSCs and PBMCs, involving cytokines, chemokines and cell death, and are of fundamental importance for tissue homeostasis.
Assuntos
Apoptose , Leucócitos Mononucleares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Cordão Umbilical/metabolismo , Quimiocina CCL2/metabolismo , Técnicas de Cocultura , Feminino , Humanos , Interferon gama/metabolismo , Interleucina-6/metabolismo , Leucócitos Mononucleares/citologia , Masculino , Células-Tronco Mesenquimais/citologia , Cordão Umbilical/citologiaRESUMO
Mesenchymal stem cells (MSCs) could be obtained from many sources, and there are differences between them. This study was purposed to compare and analyze the basic biological characteristics of umbilical cord, adipose tissue-and bone marrow-derived MSC (UC-MSCs, AD-MSCs and BM-MSCs). The MSCs were isolated from umbilical cord, adipose tissue and bone marrow were cultured; the morphology of UC-MSCs, AD-MSCs and BM-MSCs was observed by using microscopy; the immunophenotype, differentiation potential and expression of peroxisome proliferation-activated receptor-γ (PPAR-γ) mRNA were detected by using flow cytometry, differentiation test (von kossais and 0:1 red O staining) and quantitative fluorescent PCR, respectively. The results showed that the UC-MSCs, AD-MSCs and BM-MSCs displayed similar morphology under confocal microscope after being stained with rhodamine phalloidin and DAPL. The immunophenotypes of these three originated cells conform to coincide with identification criterion for MSCs, and showed similar expression level. During adipogenic induction the adipogenic potential of these MSCs was different, AD-MSCs exhibited the highest adipogenic potential, UC-MSCs displayed the lowest, while potential of BM-MSCs get between; however, the osteogenic differentiation potential of UC-MSCs, AD-MSCs and BM-MSCs was similar. The PCR detection showed that the expression level of PPAR-γ mRNA was the highest in AD-MSCs and the lowest in UC-MSCs, while expression level in BM-MSCs get between, these results were identical with the adipogenic potential, suggest that the difference of adipogenic potential in 3 kinds of MSCs was associated with basic expression level of PPAR-γ mRNA. It is concluded that UC-MSCs, AD-MSCs and BM-MSCs exhibit similar morphology, the immunophenotypes of these MSCs coincide with identification criterion for MSCs, the osteogenic potential of these MSCs is similar, while the adipogenic potential and the expression level of PPAR-γ mRNA are different. The difference-associated mechanisms need to further study.
Assuntos
Adipogenia , Tecido Adiposo/citologia , Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Cordão Umbilical/citologia , Separação Celular , Células Cultivadas , HumanosRESUMO
This study was aimed to investigate the effects of rapamycin on biological function and autophagy of bone marrow mesenchymal stem cells (BM-MSC) from patients with aplastic anemia so as to provide experimental basis for the clinical treatment of aplastic anemia (AA) with rapamycin. BM-MSC were treated with different concentrations of rapamycin (0, 10, 50, 100 nmol/L) for 48 h, the expression of LC3B protein was detected by Western blot to observe the effect of rapamycin on cell autophagy; cell apoptosis and cell cycles were detected by flow cytometry; the proliferation of BM-MSC of AA patients was measured by cell counting kit-8; the adipogenic differentiation of BM-MSC were tested by oil red O staining after adipogenic induction for 2 weeks; the adipogenic related genes (LPL, CFD, PPARγ) were detected by real-time PCR. The results showed that the proliferation and adipogenesis of BM-MSC of AA patients were inhibited by rapamycin. Moreover, the autophagy and apoptosis of BM-MSC were increased by rapamycin in a dose-dependent way.Rapamycin arrested the BM-MSC in G0/G1 phase and prevented them into S phase (P < 0.05). It is concluded that rapamycin plays an critical role in inhibiting cell proliferation, cell cycles, and adipogenesis, these effects may be related with the autophagy activation and mTOR inhibition resulting from rapamycin.
Assuntos
Anemia Aplástica/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Sirolimo/farmacologia , Apoptose/efeitos dos fármacos , Autofagia , Células da Medula Óssea/citologia , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/citologia , Transdução de SinaisRESUMO
This study was aimed to explore the immunoregulatory function and capability supporting the angiogenesis of exosomes secreted by bone marrow mesenchymal stem cells (BMMSC) from healthy persons. Supernatant of BMMSC (P4-P6) was collected for exosome purification. Transmission electron microscopy (TEM) and Western blot were used to identify the quality of isolated exosomes. The amount of exosomes was quantified through bicinchoninic acid (BCA) protein assay. Human peripheral blood mononuclear cells (PBMNC) were isolated from healthy donor and added with isolating exosomes. After co-cultured for 72 h, IFN-γ from the co-culture system was detected by ELISA. The expression of miRNA-associated with immunity were detected by real-time reverse transcription polymerase chain reaction (Real-time RT-PCR). The interactions between exosomes and human umbilical vein endothelial cells (HUVEC) were observed with confocal microscopy. Subconfluent HUVEC were harvested and treated with the indicated concentration of exosomes. Nude mice were injected subcutaneously with exosomes or PBS as control to verify the ability of angiogenesis. The results showed that diameter range of exosomes was range from 40 to 160 nm. The isolated exosomes expressed the CD9. There was approximately linear relation between the secretion of exosomes and cell density. The exosomes suppressed the production of IFN-γ from PBMNC, and contained miRNA associated with immune regulation such as miR301, miR22 and miR-let-7a. Exosomes induced vascular tube formation in vitro and vascularization of Matrigel plugs in vivo. It is concluded that the BMMSC-derived exosomes can regulate immunity and support vascularization.
Assuntos
Células da Medula Óssea/metabolismo , Exossomos/imunologia , Exossomos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Adulto , Animais , Células da Medula Óssea/citologia , Células Cultivadas , Feminino , Humanos , Interferon gama/metabolismo , Leucócitos Mononucleares/citologia , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos Nus , Pessoa de Meia-Idade , Neovascularização FisiológicaRESUMO
15-Deoxy-Δ(12), 14-prostaglandin J2 (15d-PGJ2), a well known peroxisome proliferator activated receptor (PPAR) γ ligand, has been shown to inhibit cellular proliferation and induce apoptosis and differentiation. However, whether 15d-PGJ2 influences the cytokines in the culture supernatant of bone marrow mesenchymal stem cells (BM-MSC) is unknown. This study was purposed to investigate the influence of 15d-PGJ2 on cytokines in the culture supernatant of BM-MSC. The fibroblast-like cells attached to the culture dish from bone marrow of healthy donors were isolated. The immunophenotype and differentiation potential of the obtained cells were detected by flow cytometry and oil red O and von kassa staining respectively to confirm that these cells were BM-MSC. Thereafter, the BM-MSC were cultured with complete medium supplemented with 10, 20, 40 and 60 µmol/L 15d-PGJ2 for 24 hours respectively. The real-time PCR was used to assay the PPARγ mRNA level, the confocal immuno fluorescence technique was used to detect the expression level of PPARγ. The results showed that the BM-MSC underwent apoptosis and got detached from the culture dish when the concentration of 15d-PGJ2 was no less than 20 µmol/L. The PPARγ mRNA level of BM-MSCs cultured with medium containing 10 µmol/L 15d-PGJ2 was higher than that cultured without 15d-PGJ2, and the difference was statistically significant (P < 0.05). The enhancement of PPARγ expression was observed after stimulated by 15d-PGJ2. The protein chip detecting the culture supernatants of BM-MSC cultured with 10 µmol/L 15d-PGJ2 or without 15d-PGJ2 for 24 hours demonstrated that expression levels of some of the cytokines varied. It is concluded that the down-regulation of TIMP-2 exists after treatment of 15d-PGJ2, which is statistical significant.
Assuntos
Células da Medula Óssea/metabolismo , Células-Tronco Mesenquimais/metabolismo , Prostaglandina D2/análogos & derivados , Adulto , Células da Medula Óssea/efeitos dos fármacos , Células Cultivadas , Meios de Cultura/química , Citocinas/metabolismo , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Prostaglandina D2/farmacologia , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Adulto JovemRESUMO
This study was purposed to investigate the impact of human umbilical cord-derived mesenchymal stem cells (hUC-MSC) on the sensitivity of HL-60 cells to therapeutic drugs so as to provide more information for exploring the regulatory effect of hUC-MSC on leukemia cells. Transwell and direct co-culture systems of HL-60 and hUC-MSC were established. The apoptosis and cell cycle of HL-60 cells were detected by flow cytometry. RT-PCR and Western blot were used to detect the mRNA and protein levels of Caspase 3, respectively. The results showed that the apoptosis of HL-60 induced by cytarabine (Ara-C) decreased significantly after direct co-cultured with hUC-MSC cycle mRNA (P < 0.05). The similar phenomenon was observed in transwell co-culture system. Cell cycle of HL-60 cells were arrested at G0/G1 phase and did not enter into S phase (P < 0.05) and the expression of Caspase-3 mRNA and protein in HL-60 cells were reduced (P < 0.05). It is concluded that hUC-MSC protected HL-60 from Arc-C induced apoptosis through regulating the cell cycle and down-regulating expression of Caspase 3 in HL-60 cells. In addition, this effect is caused by the soluble factors from hUC-MSC.
Assuntos
Citarabina/farmacologia , Células-Tronco Mesenquimais/citologia , Cordão Umbilical/citologia , Apoptose , Caspase 3/metabolismo , Técnicas de Cocultura , Células HL-60 , HumanosRESUMO
The aim of this study was to investigate the effects of interferon (IFN)-γ on biological characteristics and immunomodulatory property of human umbilical cord-derived mesenchymal stem cells (hUC-MSC). hUC-MSC were treated with IFN-γ 10 ng/ml (IFN-γ group) or without IFN-γ (control group). The phenotype of hUC-MSC was detected by flow cytometry. The proliferation status was detected by CCK-8 method, and its differentiation ability was assessed by oil red O and von Kossa staining. The production of PGE-2 was measured by ELISA, and the mRNA expression levels of COX-2, IDO-1 and IDO-2 in hUC-MSC were detected by real-time quantitative PCR. Furthermore, the proliferation of human peripheral blood mononuclear cells (hPBMNC) was evaluated after co-culture with hUC-MSC, IFN-γ pretreatment or not. The results showed that after IFN-γ stimulation, the expression of SSEA-4 on hUC-MSC decreased significantly [(8.15 ± 2.94) vs (16.42 ± 8.5), P < 0.05], and the expression of CD54 increased [(96.64 ± 3.29) vs (84.12 ± 10.73), P = 0.051]. The immunomodulatory property of hUC-MSC on the proliferation of hPBMNC was enhanced (P < 0.05). All the above mentioned effects were IFN-γ concentration-dependent. When hUC-MSC were stimulated by IFN-γ for 24 h, the production of PGE-2 secreted by hUC-MSC decreased significantly (P < 0.01). The mRNA expression level of COX-2 also decreased though the difference did not reach to statistically significant level. Compared with control group, IDO-1 expression level in IFN-γ group increased significantly (P < 0.01), and the mRNA expression level of IDO-2 remained unchanged. It is concluded that IFN-γ can influence the phenotype of hUC-MSC and enhance the immunomodulatory property of hUC-MSC.
Assuntos
Interferon gama/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/imunologia , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Citometria de Fluxo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Células-Tronco Mesenquimais/citologia , Cordão Umbilical/citologiaRESUMO
OBJECTIVE: Class IIa histone deacetylases (HDACs) belong to a large family of enzymes involved in protein deacetylation and play a role in regulating gene expression and cell differentiation. Previously, we showed that HDAC inhibitors modify the timing and determination of pancreatic cell fate. The aim of this study was to determine the role of class IIa HDACs in pancreas development. RESEARCH DESIGN AND METHODS: We took a genetic approach and analyzed the pancreatic phenotype of mice lacking HDAC4, -5, and -9. We also developed a novel method of lentiviral infection of pancreatic explants and performed gain-of-function experiments. RESULTS: We show that class IIa HDAC4, -5, and -9 have an unexpected restricted expression in the endocrine ß- and δ-cells of the pancreas. Analyses of the pancreas of class IIa HDAC mutant mice revealed an increased pool of insulin-producing ß-cells in Hdac5(-/-) and Hdac9(-/-) mice and an increased pool of somatostatin-producing δ-cells in Hdac4(-/-) and Hdac5(-/-) mice. Conversely, HDAC4 and HDAC5 overexpression showed a decreased pool of insulin-producing ß-cells and somatostatin-producing δ-cells. Finally, treatment of pancreatic explants with the selective class IIa HDAC inhibitor MC1568 enhances expression of Pax4, a key factor required for proper ß-and δ-cell differentiation and amplifies endocrine ß- and δ-cells. CONCLUSIONS: We conclude that HDAC4, -5, and -9 are key regulators to control the pancreatic ß/δ-cell lineage. These results highlight the epigenetic mechanisms underlying the regulation of endocrine cell development and suggest new strategies for ß-cell differentiation-based therapies.
Assuntos
Histona Desacetilases/metabolismo , Células Secretoras de Insulina/metabolismo , Pâncreas/crescimento & desenvolvimento , Proteínas Repressoras/metabolismo , Células Secretoras de Somatostatina/metabolismo , Algoritmos , Animais , Animais Recém-Nascidos , Diferenciação Celular/efeitos dos fármacos , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Histona Desacetilases/química , Histona Desacetilases/genética , Proteínas de Homeodomínio/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Camundongos Mutantes , Tamanho do Órgão , Especificidade de Órgãos , Fatores de Transcrição Box Pareados/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , RNA Mensageiro/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Somatostatina/metabolismo , Células Secretoras de Somatostatina/citologia , Células Secretoras de Somatostatina/efeitos dos fármacos , Técnicas de Cultura de TecidosRESUMO
Hematopoietic stem cells (HSC) shift from fetal liver and spleen to bone marrow at neonatal stages and this movement may be due to inductive signals from different microenvironments. Mesenchymal stem cells (MSC) are the precursors of stromal cells in bone marrow microenvironments such as osteoblasts and endothelial cells. Some researchers speculated that fetal bone marrow before birth might be not perfectly suit HSC growth. However, it is still lack of direct evidence to prove this hypothesis. This study was aimed to compare the hematopoietic supportive capacity between human fetal and adult bone marrow MSC in vitro. Adult bone marrow MSC (ABM-MSC) were isolated from three healthy donors and fetal bone marrow MSC (FBM-MSC) were isolated from three fetuses between gestations of 19 to 20 weeks. After irradiation, MSC were co-cultured with CD34(+) cells isolated from umbilical cord blood in long-term culture-initiating cell (LTC-IC) assay. The colony number of colony forming cells (CFC) was counted and the phenotypic changes of co-cultured CD34(+) cells were analyzed by flow cytometry. Cytokine expressions in both kinds of MSC were detected by reverse transcription polymerase chain reaction (RT-PCR). The results showed that ABM-MSC had a stronger hematopoietic supportive capacity than FBM-MSC. Both of them enhanced the differentiation of CD34(+) cells into myeloid lineages. Cytokines were expressed differently in ABM-MSC and FBM-MSC. It is concluded that ABM-MSC possess more potential application in some treatments than FBM-MSC, especially in hematopoietic reconstitution.
Assuntos
Células-Tronco Adultas/citologia , Células da Medula Óssea/citologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Mesenquimais/citologia , Diferenciação Celular , Células Cultivadas , Feto , Citometria de Fluxo , HumanosRESUMO
Umbilical cord mesenchymal stem cell (UCMSC) transplantation has been widely used in the treatment of a variety of diseases due to their advantages such as abundant resources, low immunogenicity and large ex vivo expansion capacity. This study was aimed to investigate the effects of UCMSC on experimental autoimmune myasthenia gravis (EAMG) rats. The distribution of human-derived cells was observed by immunofluorescence method, the effect of MSC on B-cell in situ-secreted antibodies was assayed by ELISPOT, the secreted IFN-γ level was detected by using Transwell test. The results showed that UCMSC were able to migrate to inflammation region and lymph nudes, moreover human-derived cells could be detected in medulla zone of lymph nudes. In vitro in situ detection of AchR specific antibody secretion revealed that the full contact of MSC with lymphnode-derived lymphocytes could effectively inhibit production of AchR antibody. Transwell test indicated that the direct contact of UCMSC with CD4 T cells could effectively decrease production of IFN-γ, which modulated the unbalance between Th1/Th2 to a certain extent. It is concluded that UCMSC can regulate the immune system by direct cell-cell contact or/and release of cytokines, which bring a new insight into knowledge about MSC-based therapy for EAMG.