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1.
Int Immunopharmacol ; 125(Pt A): 111091, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37883814

RESUMO

The MPLW515L mutation is a prevalent genetic mutation in patients with myeloproliferative neoplasms (MPN), and utilizing this mutation in mice model can provide important insights into the disease. However, the relationship between intestinal homeostasis and MPN mice model remains elusive. In this study, we utilized a retroviral vector to transfect hematopoietic stem cells with the MPLW515L mutation, creating mutated MPN mice model to investigate their intestinal status. Our results revealed that the MPLW515L in MPN mice model aggravated inflammation in the intestines, decreased the levels of tight junction proteins and receptors for bacteria metabolites. Additionally, there was increased activation of the caspase1/IL-1ß signaling pathway and a significant reduction in phos-p38 levels in the intestinal tissue in MPN mice. The MPLW515L mutation also led to up-expression of anti-microbial genes in the intestinal tract. Though the mutation had no impact on the alpha diversity and dominant bacterial taxa, it did influence the rare bacterial taxa/sub-communities and consequently impacted intestinal homeostasis. Our findings demonstrate the significance of MPLW515L mice model for studying MPN disease and highlight the mutation's influence on intestinal homeostasis, including inflammation, activation of the IL-1ß signaling pathway, and the composition of gut microbial communities.


Assuntos
Transtornos Mieloproliferativos , Neoplasias , Humanos , Animais , Camundongos , Mutação , Transdução de Sinais , Modelos Animais de Doenças , Janus Quinase 2/metabolismo , Inflamação , Calreticulina/genética , Calreticulina/metabolismo , Receptores de Trombopoetina
2.
Exp Hematol ; 127: 59-69.e2, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37741606

RESUMO

Most thrombopoietin receptor (MPL) mutations result in abnormal megakaryocyte expansion in the spleen or bone marrow (BM), leading to progressive fibrosis. It has been reported that p21 (Rac Family Small GTPase 1 [RAC1])-activated kinase 1 (PAK1) participates in the proliferation and differentiation of megakaryoblasts. PAK1 phosphorylation increased in patients with myeloproliferative neoplasms (MPNs) and murine MPN cells with the Mplw515l mutant gene in this study; however, the function of overactivated PAK1 in MPN cells remains unclear. We found that inhibition of PAK1 caused significant changes in the biological behaviors of MPLW515L mutant cells in vitro, including arrested growth or reduced clonality and increased polyploid DNA and cell apoptosis due to upregulated cleaved caspase 3. In vivo, PAK1 inhibitor treatment caused a slow elevation of leukocytosis and hematocrit (HCT) and a reduction in hepatosplenomegaly in 6133/MPLW515L-transplanted mice, along with reduced tumor cell infiltration and prolonged survival. Further, deletion of PAK1 sustained a relatively normal HCT and platelet count at the beginning of the disease but did not completely alleviate the splenomegaly of MPLW515L mutant mice. Notably, PAK1 knockout attenuated the destruction of splenic structure, and reduced the megakaryocyte burden within the BM. These results suggest that inhibition of PAK1 may be a useful method for treating MPLW515L mutant MPN by intervening megakaryocytes.


Assuntos
Transtornos Mieloproliferativos , Neoplasias , Humanos , Camundongos , Animais , Megacariócitos/patologia , Proliferação de Células , Neoplasias/patologia , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Diferenciação Celular , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/farmacologia
3.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 31(4): 1113-1118, 2023 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-37551485

RESUMO

OBJECTIVE: To construct a myeloproliferative neoplasms (MPN) transplanted mouse model with JAK2-V617F, MPLW515L or CALR-Type I gene mutation, and establish a systematic evaluation system to verify the success of model construction. METHODS: The bone marrow c-kit+ cells of the mice were obtained by the following steps: The mice were killed by cervical dislocation, the femur, tibia and ilium were separated, and the bone marrow cells were collected. The c-kit+ cells were sorted after incubation with CD117 magnetic beads. The method of constructing mouse primary mutant cells is as follows: A gene mutation vector with a GFP tag was constructed by the retroviral system, and the retroviral vector was packaged into the Platinum-E cells to obtain the virus supernatant, and then used it to infect the c-kit+ cells of mice. The MPN mouse model was constructed as follows: the mouse primary c-kit+ cells containing the mutant genes were collected after infection, and then transplanted them via the tail vein into the female recipient mice of the same species which were irradiated with a lethal dose of gamma rays (8.0 Gy). The MPN mouse model was evaluated as follows: After transplantation, the peripheral blood of the mice was regularly collected from the tail vein to perform the complete blood count test, and the size of spleen and the degree of bone marrow fibrosis were estimated. RESULTS: The mouse c-kit+ cells with the mutant genes were successfully obtained from the bone marrow. MPN mouse model was successfully constructed: The peripheral blood cells of the MPN-transplanted mice carried exogenous implanted GFP-positive cells, and the white blood cells (WBC), platelet (PLT) and hematocrit (HCT) were all increased; the body weight loss, and the water and food intake were reduced in the transplanted mice; further pathological analysis showed that the transplanted mice displayed splenomegaly and bone marrow fibrosis. These results suggested that the MPN mouse model was successfully constructed. According to the common and different characteristics of the three MPN mouse model, a preliminary evaluation system for judging the success of MPN mouse model construction was summarized, which mainly included the following indicators, for example, the proportion of GFP-positive cells in the peripheral blood of mice; WBC, PLT and HCT; the degree of spleen enlargement and the bone marrow fibrosis. CONCLUSION: The MPN mouse model with JAK2-V617F, MPLW515L or CALR-Type I gene mutation is successfully established by retroviral system, which can provide an important experimental animal model for the research of MPN pathogenesis and drug-targeted therapy.


Assuntos
Transtornos Mieloproliferativos , Neoplasias , Mielofibrose Primária , Feminino , Camundongos , Animais , Transtornos Mieloproliferativos/genética , Medula Óssea/patologia , Mutação , Modelos Animais de Doenças , Janus Quinase 2/genética
4.
Leuk Res ; 120: 106920, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35872339

RESUMO

Acute megakaryoblastic leukaemia (AMKL) is characterized by expansion of megakaryoblasts, which are hyper-proliferative cells that fail to undergo differentiation. Insight to the cell-cycle regulation revealed important events in early or late megakaryocytes (MKs) maturation; the cyclin-dependent kinases 4 and 6 (CDK4/6) have been reported to participate in the development of progenitor megakaryocytes, mainly by promoting cell cycle progression and DNA polyploidization. However, it remains unclear whether the continuous proliferation, but not differentiation, of megakaryoblasts is related to an aberrant regulation of CDK4/6 in AMKL. Here, we found that CDK4/6 were up regulated in patients with AMKL, and persistently maintained at a high level during the differentiation of abnormal megakaryocytes in vitro, according to a database and western blot. Additionally, AMKL cells were exceptionally reliant on the cell cycle regulators CDK4 or 6, as blocking their activity using an inhibitor or short hairpin RNA (shRNA) significantly reduced the proliferation of 6133/MPL megakaryocytes, reduced DNA polyploidy, induced apoptosis, decreased the level of phosphorylated retinoblastoma protein (p-Rb), and activation of caspase 3. Additionally, CDK4/6 inhibitors and shRNA reduced the numbers of leukemia cells in the liver and bone marrow (BM), alleviated hepatosplenomegaly, and prolonged the survival of AMKL-transplanted mice. These results suggested that blocking the activity of CDK4/6 may represent an effective approach to control megakaryoblasts in AMKL.


Assuntos
Leucemia Megacarioblástica Aguda , Animais , Ciclinas , DNA , Leucemia Megacarioblástica Aguda/tratamento farmacológico , Leucemia Megacarioblástica Aguda/genética , Células Progenitoras de Megacariócitos , Camundongos , RNA Interferente Pequeno
5.
Shanghai Kou Qiang Yi Xue ; 23(1): 7-14, 2014 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-24608605

RESUMO

PURPOSE: To study the feasibility of tissue engineered bone constructed with simvastatin carried by PLGA/CPC and bone marrow stromal cells (BMSCs) and screen the effective drug loading of simvastatin. METHODS: Solvent casting-particle leaching technology combined with the phase separation process was used to prepare the different concentrations (simvastatin mass: 0.1, 0.5, 1 mg) of simvastatin carried by PLGA/CPC composite scaffold materials. Scanning electron microscopy was used to observe the porosity and drug release curve was drawn; Alizarin red staining and type I collagen staining were applied to observe the effect of osteogenic medium and simvastatin on the role of BMSCs to the osteogenetic differentiation. The induced passage 3 cells after dil staining were mixed with the composite scaffold material to a complex. Scanning electron microscopy and laser confocal microscope were used to observe the adhesion on the complex. CCK-8 and alkaline phosphatase (ALP) were applied to observe the proliferation and differentiation. SPSS 18.0 software package was used for statistical analysis. RESULTS: The scaffold porosity was more than 90% with an average aperture of 200-300 µm. The drug released slowly. There was no obvious interpretation. Type I collagen showed positive expression. Alizarin red staining proofed the formation of mineralization nodules in group which was induced with the conditional medium and simvastatin. 0.5 mg group showed cells adhered to the inner surface of the scaffold material and could significantly promote the proliferation and differentiation of cells. CONCLUSIONS: Combination of simvastatin and osteogenic medium can effectively promote the differentiation of BMSCs. Simvastatin carried by PLGA/CPC scaffold materials is an ideal tissue engineering scaffold material. PLGA/CPC scaffold containing 0.5 mg simvastatin can effectively promote the proliferation and differentiation of BMSCs. Supported by Natural Science Foundation of Jilin Province (201215052).


Assuntos
Células-Tronco Mesenquimais , Sinvastatina , Fosfatase Alcalina , Células da Medula Óssea , Fosfatos de Cálcio , Diferenciação Celular , Ácido Láctico , Osteogênese , Ácido Poliglicólico/análogos & derivados , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Engenharia Tecidual , Alicerces Teciduais
6.
J Cardiovasc Med (Hagerstown) ; 15(7): 579-86, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24583668

RESUMO

OBJECTIVES: A great number of studies indicate that cardiac fibroblasts are essential for maintaining the structure and function of heart. Hypoxia-inducible factor-1 alpha (HIF-1α) is a central transcriptional regulator of hypoxic response. The present study examined whether over-expression of HIF-1α could prevent hypoxia-induced injury in neonatal rat cardiac fibroblasts and, if so, its possible molecular targets. METHODS: Western blotting was used to detect protein level. MTT, electron microscopy, TUNEL staining and confocal microscopy were used to identify cell viability, cell apoptosis and intracellular calcium ([Ca]i) in cardiac fibroblasts, respectively. RESULTS: When cardiac fibroblasts were exposed to hypoxia, HIF-1α protein in nuclei was transiently accumulated at 1 h, and then gradually degraded within 24 h of hypoxia exposure. Over-expression of HIF-1α enhanced nucleus expression of HIF-1α in cardiac fibroblasts, and significantly abolished the decrease of cell viability and cell apoptosis caused by 24-h hypoxia. Accordingly, hypoxia-induced Bax up-regulation, Bcl-2 down-regulation, caspase-3 activation and overload of [Ca]i in cardiac fibroblasts were reversed by HIF-1α over-expression, but were promoted by 30 µmol/l SC205346, a specific HIF-1α blocker. CONCLUSIONS: Our results indicate that HIF-1α may act as a protective factor in the apoptotic process of cardiac fibroblasts and represent a potential therapeutic target for heart remodeling after hypoxia injury.


Assuntos
Apoptose/genética , DNA/genética , Fibroblastos/ultraestrutura , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Isquemia Miocárdica/genética , Miócitos Cardíacos/ultraestrutura , Animais , Western Blotting , Cálcio/metabolismo , Caspase 3/metabolismo , Sobrevivência Celular , Ensaio de Imunoadsorção Enzimática , Fibroblastos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Marcação In Situ das Extremidades Cortadas , Microscopia Confocal , Microscopia Eletrônica , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Miócitos Cardíacos/metabolismo , Ratos , Transdução de Sinais
7.
Toxicol Lett ; 219(3): 223-30, 2013 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-23542815

RESUMO

Arsenic trioxide (As2O3; ATO) is clinically effective in treating acute promyelocytic leukemia (APL); however, it frequently causes cardiotoxic effects. This study was designed to investigate whether ATO could induce apoptosis of cardiac fibroblasts (CFs) that play very important roles in maintaining the structure integrity and function of the heart. Cardiac fibroblasts from guinea pigs administered with ATO (1mg/kgbw) were used to test the pro-apoptotic role of ATO in vivo. The current study demonstrated that ATO induced morphological characteristics of apoptosis and Caspase-3 activation in CFs of guinea pigs along with a significant up-regulation in TGF-ß1 protein expression, Bax/Bcl-2 ratio and ERK1/2 phosphorylation. In vitro MTT assay showed that ATO remarkably reduced the viability of cultured cardiac fibroblasts (NRCFs) from neonatal rat in a concentration- and time-dependent manner. Consistent with the notions in vivo, ATO significantly induced the apoptosis in NRCFs, dramatically up-regulated TGF-ß1 protein level and Bax/Bcl-2 ratio in a time-dependent fashion and activated Caspase-3 and ERK1/2. Finally, pretreatment with LY364947, an inhibitor of TGF-ß signaling could apparently reverse these changes. We therefore conclude that TGF-ß is functionally linked to ERK1/2 and that TGF-ß signaling is responsible for ATO-induced CFs apoptosis, which provides a novel mechanism of ATO related cardiac toxicology.


Assuntos
Apoptose/efeitos dos fármacos , Arsenicais/farmacologia , Fibroblastos/efeitos dos fármacos , Coração/efeitos dos fármacos , Óxidos/farmacologia , Fator de Crescimento Transformador beta1/biossíntese , Animais , Trióxido de Arsênio , Western Blotting , Caspase 3/metabolismo , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Cobaias , Masculino , Microscopia Eletrônica , Miocárdio/citologia , Miocárdio/metabolismo , Ratos , Regulação para Cima/efeitos dos fármacos
8.
Cardiovasc Res ; 96(1): 90-8, 2012 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-22853924

RESUMO

AIMS: Arsenic trioxide (ATO), an effective therapeutic agent for acute promyelocytic leukaemia, can cause sudden cardiac death due to long QT syndrome (LQTS). The present study was designed to determine whether ATO could induce cardiac fibrosis and explore whether cardiac fibroblasts (CFs) are involved in the development of LQTS by ATO. METHODS AND RESULTS: ATO treatment of guinea pigs caused substantial interstitial myocardial fibrosis and LQTS, which was accompanied by an increase in transforming growth factor ß1(TGF-ß1) secretion and a decrease in ether-à-go-go-related gene (HERG) and inward rectifying potassium channel (I(K1)) subunit Kir2.1 protein levels. ATO promoted collagen production and TGF-ß1 expression and secretion in cultured CFs. Whole-cell patch clamp and western blotting showed that treatment with TGF-ß1 markedly reduced HERG and I(K1) current densities and downregulated HERG and Kir2.1 protein expression in HEK293 cells stably transfected with the human recombinant HERG channel and in cardiomyocytes (CMs). These changes were completely reversed by treatment with the protein kinase A (PKA) antagonist, H89. CM and CF co-cultures showed that ATO significantly increased TGF-ß1 levels in the culture medium, whereas markedly reduced HERG and Kir2.1 protein levels were observed in CMs compared with ATO-treated CMs not co-cultured with CFs. Finally, in vivo administration of LY364947, a pharmacological antagonist of TGF-ß signalling, dramatically prevented interstitial fibrosis and LQTS and abolished aberrant expression of TGF-ß1, HERG, and Kir2.1 in ATO-treated guinea pigs. CONCLUSION: ATO-induced TGF-ß1 secretion from CFs aggravates QT prolongation, suggesting that modulation of TGF-ß signalling may provide a novel strategy for the treatment of drug-induced LQTS.


Assuntos
Antineoplásicos/efeitos adversos , Arsenicais/efeitos adversos , Fibroblastos/metabolismo , Síndrome do QT Longo/induzido quimicamente , Óxidos/efeitos adversos , Fator de Crescimento Transformador beta1/metabolismo , Animais , Animais Recém-Nascidos , Trióxido de Arsênio , Colágeno/metabolismo , Regulação para Baixo , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/metabolismo , Fibrose , Cobaias , Células HEK293 , Humanos , Síndrome do QT Longo/metabolismo , Síndrome do QT Longo/patologia , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais
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