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Many reports have presented that in tight formation, the flow mechanism differs from a conventional reservoir, such as molecular diffusion, Pre-Darcy flow behavior, and stress sensitivity. However, for CO2 Huff-n-Puff development, it is a challenge to synthetically research these mechanisms. Considering the above flow mechanisms and offshore engineering background, the development plan optimization becomes a key issue. In this paper, a self-developed simulator that satisfies research needs is introduced. Then, based on experimental results, the simulation is launched to analyze the effects of CO2 diffusion, Huff-n-Puff period, and permeability heterogeneity. The results indicate that molecular diffusion makes a positive contribution to the oil recovery factor. Additionally, for offshore reservoirs, limited to the development cost and CO2 facilities corrosion, when the total Huff-n-Puff time is constant, the ratio of 0.5-1.0 between the Huff period and the Puff period in every cycle performs better. Finally, the greater heterogeneity in permeability is much more favorable for the CO2 Huff-n-Puff because of more intensive transport processes in formation. These different scenarios can increase the understanding of the CO2 Huff-n-Puff in tight oil offshore reservoirs.
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A facile tandem oxa-Nazarov cyclization and dibromination has been developed. The combination of Cu(OTf)2 and diphenyl phosphate (DPP-H) was found to synergistically promote the coupling of conjugated 1,2-diketones and N-bromosuccinimide to form 2,4-dibromo-3(2H)-furanones in good yields.
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Cardiovascular diseases are the leading causes of mortality and morbidity worldwide. Atherosclerotic plaque underlies the predominant factors and is composed of various cell types, including structure cells, such as endothelial and smooth muscle cells, and immune cells, such as macrophages and T cells. Single-cell RNA sequencing (scRNA-seq) has been extensively applied to decipher these cellular heterogeneities to expand our understanding on the mechanisms of atherosclerosis (AS) and to facilitate identifying cell-type-specific long noncoding RNAs (LncRNAs). LncRNAs have been demonstrated to deeply regulate biological activities at the transcriptional and post-transcriptional levels. A group of well-documented functional lncRNAs in AS have been studied. In our review, we selectively described several lncRNAs involved in the critical process of AS. We highlighted four novel lncRNAs (lncRNA CARMN, LINC00607, PCAT19, LINC01235) detected in scRNA-seq datasets and their functions in AS. We also reviewed open web source and bioinformatic tools, as well as the latest methods to perform an in-depth study of lncRNAs. It is fundamental to annotate functional lncRNAs in the various biological activities of AS, as lncRNAs may represent promising targets in the future for treatment and diagnosis in clinical practice.
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Aterosclerose , Doenças Cardiovasculares , Placa Aterosclerótica , RNA Longo não Codificante , Humanos , RNA Longo não Codificante/metabolismo , Aterosclerose/metabolismo , Placa Aterosclerótica/metabolismo , Macrófagos/metabolismo , Doenças Cardiovasculares/metabolismoRESUMO
Chemical looping combustion (CLC) is a potential CO2 capture and sequestration (CCS) technology that can easily separate CO2 and H2O without energy loss and greatly improve the efficiency of carbon capture. Due to the inherent defects of natural iron ore, such as low reactivity and poor oxygen carrying capacity, four kinds of biomass ashes (rape stalk ash, rice stalk ash, platane wood ash, and U. lactuca ash) that have different constituents of K, Na, Ca, and Si were applied to modify the redox performance of natural iron ore. The effects of biomass ash type, constituent, reaction temperature, H2O vapor flow rate, and redox cycle on the CLC process were assessed experimentally in a batch fluidized bed reactor system. Oxygen carrier physicochemical characteristics were determined by several analytical techniques. The results showed that rape stalk ash, platane wood ash, and U. lactuca ash with a high K content and high K/Si ratio significantly improved the reactivity and cycle stability of iron ore, even after 10 redox cycles, while rice straw ash with a low K/Si ratio showed an inhibitory effect due to the formation of bridge eutectics, which enhanced agglomeration. In a range from 800 to 950 °C, higher temperatures led to a much better ability to promote the CLC process than lower temperatures. A higher flow rate of H2O had little effect on the further promotion of the CLC process due to hydrogen inhibition. It is believed that the application of BA-modified iron ore oxygen carriers is an effective strategy to improve the CLC process.
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BACKGROUND: Mammalian target of rapamycin (mTOR) is crucial for endothelial function. This study is aimed at assessing whether the glucagon-like peptide-1 (GLP-1) analogue liraglutide has a protective effect on endothelial function via the mTOR signaling pathway. METHODS: Human umbilical vein endothelial cells (HUVECs) were administered liraglutide (100 nM) for 0, 10, 30, 60, 720, and 1440 minutes, respectively. Then, the expression and phosphorylation levels of mTOR, mTOR-Raptor complex (mTORC1), and mTOR-Rictor complex (mTORC2) were determined by Western blot and immunoprecipitation, while mTORC1 and mTORC2 expression was blocked by siRNA-Raptor and siRNA-Rictor, respectively. Akt phosphorylation was detected by Western blot. HUVECs were then incubated with liraglutide in the absence or presence of Akt inhibitor IV. Nitric oxide (NO) release was assessed by the nitrate reductase method. Phosphorylated endothelial nitric oxide synthase (eNOS), human telomerase reverse transcriptase (hTERT), and apoptosis-related effectors were assessed for protein levels by Western blot. Telomerase activity was evaluated by ELISA. RESULTS: Sustained mTOR phosphorylation, mTORC2 formation, and mTORC2-dependent Akt phosphorylation were induced by liraglutide. In addition, eNOS phosphorylation, NO production, nuclear hTERT accumulation, and nuclear telomerase activity were enhanced by mTORC2-mediated Akt activation. Liraglutide also showed an antiapoptotic effect by upregulating antiapoptotic proteins and downregulating proapoptotic proteins in an mTORC2-Akt activation-dependent manner. CONCLUSION: Liraglutide significantly improves endothelial function, at least partially via the mTORC2/Akt signaling pathway.
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Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Incretinas/farmacologia , Liraglutida/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
Resistance to cytarabine is an important cause of therapy failure in persons with acute myeloid leukemia (AML). Deoxycytidine kinase, encoded by DCK, catalyzes phosphorylation of cytarabine to cytarabine monophosphate, a necessary step for eventual incorporation of cytarabine triphosphate into DNA and for clinical efficacy. Whether DCK mutations make AML cells resistant to cytarabine is controversial. We studied DCK mutations and messenger RNA (mRNA) concentrations in leukemia cells from 10 subjects with AML who received cytarabine-based therapy and relapsed and in 2 artificially induced cytarabine-resistant AML cell lines. DCK mutations were detected in 4 subjects with AML relapsing after achieving a complete remission and receiving high-dose cytarabine postremission therapy. Most mutations were in exons 4-6 and were not present before therapy. DCK was also mutated in cytarabine-resistant but not parental AML cell lines. DCK mRNA concentrations were significantly decreased in cytarabine-resistant K562 and SHI-1 cells compared with cytarabine-sensitive parental cells. Mutation frequency of DCK and mRNA concentration did not correlate with the extent of cytarabine resistance indicating other factors operate. Overexpression of wild-type DCK restored cytarabine sensitivity to previously resistant leukemia cell lines. Our data contribute to the understanding of cytarabine resistance in persons with AML.
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Citarabina/farmacologia , Desoxicitidina Quinase , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Mieloide Aguda , Mutação , Proteínas de Neoplasias , Desoxicitidina Quinase/genética , Desoxicitidina Quinase/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Células K562 , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismoRESUMO
Although the topography of mutations in persons of predominately European-descent with acute myeloid leukemia (AML) is well-described this is less so in Asians. We studied AML-related mutations in 289 consecutive Chinese (mostly Han) with newly-diagnosed de novo AML. Full-length coding sequence of NPM1 and CEBPA, IDH1 and IDH2 hotspot mutations and WT1 mutations in exons 7 and 9 were analyzed by PCR as were correlations with clinical and laboratory variables. CEBPA mutations were detected in 20% of subjects (95% confidence interval [CI] 15, 25%), NPM1 mutations in 20% (15, 25%), IDH1 mutations in 4% (1, 6%), IDH2 mutations in 11% (7, 15%) and WT1 mutations in 6% (3, 9%). A comparison of these data with mutation frequencies in persons of predominately European-descent with AML indicates a higher frequency of CEBPA mutations, a similar frequency of IDH2 mutations and lower frequencies of NPM1, IDH1 and WT1 mutations. Our data indicate different topographies of AML-associated mutations in Chinese compared with persons of predominately European descent suggesting genetic background, life-style, environment and perhaps other variables may influence these differences.
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Povo Asiático/genética , Biomarcadores Tumorais , Leucemia Mieloide Aguda/genética , Mutação , População Branca/genética , Adolescente , Adulto , Idoso , Criança , Aberrações Cromossômicas , Feminino , Humanos , Leucemia Mieloide Aguda/diagnóstico , Masculino , Pessoa de Meia-Idade , Nucleofosmina , Adulto JovemRESUMO
Co-pyrolysis characteristics of petrochemical wastewater sludge and Huolinhe lignite were investigated using thermogravimetric analyzer and packed-bed reactor coupled with Fourier transform infrared spectrometer and gas chromatography. The pyrolysis characteristics of the blends at various sludge blending ratios were compared with those of the individual materials. Thermogravimetric experiments showed that the interactions between the blends were beneficial to generate more residues. In packed-bed reactor, synergetic effects promoted the release of gas products and left less liquid and solid products than those calculated by additive manner. Fourier transform infrared spectrometer analysis showed that main functional groups in chars gradually disappeared with pyrolysis temperatures increasing, and H2O, CH4, CO, and CO2 appeared in volatiles during pyrolysis. Gas compositions analysis indicated that, the yields of H2 and CO clearly increased as the pyrolysis temperature and sludge blending ratio increasing, while the changes of CH4 and CO2 yields were relatively complex.
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Reatores Biológicos , Esgotos/química , Purificação da Água/métodos , Carvão Mineral/análise , Temperatura Alta , CinéticaRESUMO
BACKGROUND: BCL11A encodes a C2H2 type zinc-finger protein. During normal haematopoietic cell differentiation BCL11A expression is down-regulated. Data in mice suggest up-regulation of BCL11A is involved in the pathogenesis of myeloid leukaemias. BCL11A expression in persons with acute myeloid leukaemia (AML) is not systematically studied. OBJECTIVE: Interrogate associations between BCL11A expression at diagnosis and clinical and laboratory valuables and outcomes in newly-diagnosed persons with AML. METHODS: We determined BCL11A mRNA levels in bone marrow and blood mononuclear cells in 292 consecutive newly-diagnosed subjects with AML by reverse transcript and real-time polymerase chain reaction. Data were compared to mRNA levels in bone marrow cells of normals. RESULTS: Subjects with BCL11A transcript levels at diagnosis exceeding the median value of 2.434 (±3.423 SD; 25th-75th inter-quartile range, 1.33-4.29) had higher WBC levels, a greater proportion of bone marrow myeloblasts, were more likely to be FAB M0 subtype, less likely to be FAB M3 subtype, more likely to be in the intermediate cytogenetic risk cohort, less likely to have a complex karyotype and more likely to have DNMT3A(R882) and FLT3-ITD mutations than subjects with transcript levels below the median value. In 89 subjects receiving conventional induction chemotherapy the complete remission rate was 54% (95% confidence interval [CI]; 33, 75%) in the lower BCL11A cohort and 65% (45, 85%; P=0.26) in the higher BCL11A cohort. 3 year survival was 33% (2, 65%) in the lower BCL11A cohort and 15% (0, 39%; P=0.35) in the high BCL11A cohort. CONCLUSION: BCL11A transcript levels at diagnosis was significantly associated with several clinical and laboratory variables. There were also non-significant associations with complete remission rate and survival. These data suggest a possible role for BCL11A expression in AML biology.
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Proteínas de Transporte/biossíntese , Leucemia Mieloide Aguda/patologia , Proteínas Nucleares/biossíntese , Adolescente , Adulto , Idoso , Proteínas de Transporte/genética , Criança , Feminino , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Proteínas Repressoras , Adulto JovemRESUMO
The pyrolysis and oxy-fuel combustion characteristics of petrochemical wastewater sludge (PS) were studied in air (O2/N2) and oxy-fuel (O2/CO2) atmospheres using non-isothermal thermogravimetric analysis (TGA). Pyrolysis experiments showed that the weight loss profiles were almost similar up to 1050K in both N2 and CO2 atmospheres, while further weight loss took place in CO2 atmosphere at higher temperatures due to char-CO2 gasification. Compared with 20%O2/80%N2, the drying and devolatilization stage of PS were delayed in 20%O2/80%CO2 due to the differences in properties of the diluting gases. In oxy-fuel combustion experiments, with O2 concentration increasing, characteristic temperatures decreased, while characteristic combustion rates and combustion performance indexes increased. Kinetic analysis of PS decomposition under various atmospheres was performed using Coats-Redfern approach. The results indicated that, with O2 concentration increasing, the activation energies of Step 1 almost kept constant, while the values of subsequent three steps increased.
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Temperatura Alta , Cinética , Esgotos/química , Termogravimetria/métodos , Águas Residuárias/química , Atmosfera , Dióxido de Carbono/farmacologia , Gases/química , Oxigênio/farmacologiaRESUMO
Pyrolysis characteristics of four distinct oil-plant wastes were investigated using TGA and fixed-bed reactor coupled with GC. TGA experiments showed that the pyrolysis behaviors were related to biomass species and heating rates. As the heating rate increased, TG and DTG curves shifted to the higher temperatures, and the comprehensive devolatilization index obviously increased. The remaining chars from TGA experiments were higher than those obtained from the fixed-bed experiments. The crack of tars at high temperatures enhanced the formation of non-condensable gases. During the pyrolysis, C-O and CO2 were the major gases. Chars FTIR showed that the functional groups of O-H, C-H(n), C=O, C-O, and C-C gradually disappeared from 400 °C on. The kinetic parameters were calculated by Coats-Redfern approach. The results manifested that the most appropriate pyrolysis mechanisms were the order reaction models. The existence of kinetic compensation effect was evident.
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Biocombustíveis , Calefação/instrumentação , Hidrogênio/química , Modelos Químicos , Eliminação de Resíduos/instrumentação , Simulação por Computador , Desenho de Equipamento , Análise de Falha de Equipamento , Hidrogênio/análise , Cinética , Extratos Vegetais , Óleos de Plantas , TemperaturaRESUMO
The pyrolysis characteristics of petrochemical wastewater sludge (PS) were evaluated using TG/DSC-FTIR and fixed-bed reactor with GC. TGA experiments indicated that the pyrolysis of PS proceeded in three phases, and the thermographs shifted to higher temperatures with increasing heating rate. Chars FTIR showed that the absorption of O-H, C-H, C=O and C-C decreased with pyrolysis temperatures increasing. Gases FTIR correspondingly showed that H2O, CO, and CH4 generated at higher temperatures. For the fixed-bed reactor tests, H2 and CO were relatively higher in the pyrolysis gases, and CH4 was negligible at 436K. The kinetic triplets of PS pyrolysis were estimated by Flynn-Wall-Ozawa, Kissinger-Akahira-Sunose, and integral master-plots method. The results suggested that the most potential kinetic models for the first and second phase were the order reaction model, while the random nucleation and nuclei growth model for the third phase.
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Esgotos/análise , Águas Residuárias/análise , Monóxido de Carbono/química , Cromatografia Gasosa/métodos , Gases/química , Calefação , Temperatura Alta , Cinética , Metano/química , Esgotos/química , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Termogravimetria/métodos , Águas Residuárias/químicaRESUMO
Co-pyrolysis behaviors of refining and chemicals wastewater solid (RS) and Huolinhe lignite (HL) were investigated via thermogravimetric analysis (TGA). The thermal degradation process of RS and the blends proceeded in three stages, while two stages for HL. The increased percentage of RS in the blends reduced the characteristic temperature (Ti, Tp, Tf) and residual mass (Mr), while raised the characteristic reaction rate (Rp, Rv) and comprehensive devolatilization parameter (D). The results indicated that there existed some inhibitive interactions between RS and HL. The activation energies were calculated by using the Friedman and Starink method. The activation energy of RS increased first and then decreased with conversion degree, and the variation wasn't as great as that of the blends and lignite. No matter which conversion degree is, the activation energy decreased with the percentage of RS in the blends increasing. The minimum value was obtained by blending 75wt.% RS.
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Carvão Mineral , Águas Residuárias/química , Cinética , Termogravimetria/métodosRESUMO
Ecotropic viral integration site-1 (EVI1) proto-oncogene expression in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) requires further investigation. Here, EVI1 expression levels were measured in 216 Chinese patients with AML and 67 with ALL via quantitative real-time polymerase chain reaction. We found that EVI1 expressed at a high level (H-EVI1) was present in 11.1% of patients with AML versus 20.9% with ALL. Low levels of EVI1 expression occurred in 23.1% with AML versus 43.3% with ALL. This suggested that alteration of EVI1 expression was more profound in ALL than in AML. H-EVI1 was significantly enriched in 30-60-year-old patients. French-American-British (FAB) M3 subtype was significantly correlated with H-EVI1. Interestingly, we found that EVI1 expression was negatively associated with presence of the Philadelphia chromosome (Ph+) and MLL rearrangements in AML. However, Ph+, but not MLL rearrangements, was inversely correlated with EVI1 expression in B-ALL. These results for the first time suggest a mutually exclusive relationship between EVI1 expression and Ph+ karyotype.
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Proteínas de Ligação a DNA/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proto-Oncogenes/genética , Fatores de Transcrição/genética , Doença Aguda , Adolescente , Adulto , Idoso , Criança , Feminino , Rearranjo Gênico , Histona-Lisina N-Metiltransferase/genética , Humanos , Cariótipo , Leucemia Promielocítica Aguda/genética , Proteína do Locus do Complexo MDS1 e EVI1 , Masculino , Pessoa de Meia-Idade , Proteína de Leucina Linfoide-Mieloide/genética , Cromossomo Filadélfia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Proto-Oncogene Mas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
In eukaryotic cells, the endoplasmic reticulum (ER) is a very critical site for synthesis, folding, modification of protein, and calcium homeostasis. The ER responds to factors that perturb ER function such as the accumulation of unfolded proteins (ER stress) by activating unfolded protein response to relieve the stress. However, chronic or unresolved ER stress can induce neuronal apoptosis by activating c-Jun N-terminal kinase (JNK), glycogen synthase kinase 3/3ß (GSK3/3ß), CAAT/enhancer binding protein homologous protein (CHOP), and caspase-12 pathway. Research related to ER stress will provide therapeutic implications in neurological diseases.
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Apoptose/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/fisiologia , Neurônios/patologia , Neurônios/fisiologia , Transdução de Sinais/fisiologia , Animais , Sobrevivência Celular/fisiologia , Humanos , Neurônios/citologiaRESUMO
Macrophage foam cells formation is the most important process in atherosclerotic plaque formation and development. Toll-like receptor 4 (TLR4) is one of the important innate immune sensors of endogenous damage signals and crucial for regulating inflammation. Growing evidence indicates that TLR4 plays a very important role in macrophage foam cells formation. However, the underlying mechanisms regulating TLR4 expression in macrophage are not fully understood. In this study, we induced THP-1 macrophage foam cells formation with oxidative modified low-density lipoprotein (ox-LDL). We observed that TLR4 mRNA and protein expression were markedly up-regulated, and the phosphorylation of mammalian target of rapamycin (mTOR) and its downstream target p70S6K were promoted during foam cells formation. The mTOR inhibitor rapamycin blocked mTOR phosphorylation and inhibited TLR4 expression induced by ox-LDL. Silencing mTOR, rictor or raptor protein expression by small interfering RNA, also inhibited the up-regulation of TLR4 expression, respectively. Inhibition of mTOR with rapamycin reversed the down-regulation of cellular lipid efflux mediator ABCA1, which resulted from the activation of TLR4 by ligands. These data suggested that TRL4 expression was up-regulated by a mechanism dependent on mTOR signal pathway activation during THP-1 macrophage foam cells formation. Inhibition of ox-LDL induced mTOR activation reduced TLR4 expression, and improved the impaired lipid efflux.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Células Espumosas/citologia , Células Espumosas/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Receptor 4 Toll-Like/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Aterosclerose/patologia , Proteínas de Transporte/genética , Ativação Enzimática , Humanos , Fosforilação , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Proteína Companheira de mTOR Insensível à Rapamicina , Proteína Regulatória Associada a mTOR , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Regulação para CimaRESUMO
OBJECTIVE: Circulating angiogenic cells (CACs) participate in neovascularization and arterial repair. Although high-density lipoprotein (HDL) is known to enhance the functional activity of CACs, the mechanisms underlying this regulation are poorly understood. Here, we examined the mechanism(s) by which reconstituted HDL (rHDL) affects CAC senescence. METHODS AND RESULTS: CACs isolated from human peripheral blood and treated with rHDL displayed reduced senescence, as measured by acidic ß-galactosidase staining. This protective effect was blocked by the mammalian target of rapamycin (mTOR) inhibitor (rapamycin). According to Western blot analysis and immunoprecipitation results, rHDL promoted mTOR phosphorylation, mTOR-rictor complex formation, and mTOR-rictor-dependent Akt activation, which were accompanied by increased nuclear translocation of human telomerase reverse transcriptase and enhanced nuclear telomerase activity. Suppression of rictor gene expression with a small interfering RNA blocked mTOR-rictor complex formation and Akt activation. The suppression also abolished the rHDL-induced inhibition of CAC senescence and promotion of nuclear telomerase activity. Treatment of aged mice with rHDL attenuated spleen-derived CAC senescence. In CACs isolated from rHDL-treated aged mice, the phosphorylated mTOR and Akt levels were significantly enhanced. CONCLUSION: rHDL stimulates sustained mTOR phosphorylation and mTOR-rictor complex formation and inhibits senescence onset in CACs through mTOR complex 2 pathway activation.
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Senescência Celular , Lipoproteínas HDL/farmacologia , Neovascularização Fisiológica , Transporte Ativo do Núcleo Celular , Animais , Células Cultivadas , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multiproteicos , Fosforilação , Proteínas/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Telomerase/metabolismoRESUMO
OBJECTIVE: To investigate the effects of rapamycin on cholesterol homeostasis and secretory function of 3T3-L1 cells. METHODS: The in vitro cultured 3T3-L1 cells (preadipocytes) were divided into control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group. Intracellular cholesterol level was measured by oil red O staining and high performance liquid chromatography. The secretion levels of leptin and adiponectin were assayed by enzyme-linked immunosorbent assay. The mRNA and protein expressions of peroxisome proliferator-activated receptor (PPARgamma) were assayed by quantitative real-time polymerase chain reaction and Western blot. RESULTS: Oil red O staining showed rapamycin down-regulated 3T3-L1 cells differentiation and lipid accumulation. Quantitative measurement of cholesterol with high performance liquid chromatography showed that the concentrations of free cholesterol in rapamycin treatment groups had a significant reduction. The concentrations of free cholesterol in the control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were (12.89 +/- 0.16), (9.84 +/- 0.45), (9.39 +/- 0.46), and (8.61 +/- 0.34) mg/ml, respectively (P < 0.05), and the concentrations of total cholesterol were (12.91 +/- 0.50), (9.94 +/- 0.96), (10.45 +/- 2.51), and (9.53 +/- 0.63) mg/ml, respectively. The leptin concentrations in the control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were (19.02 +/- 0.52), (16.98 +/- 0.11), (15.62 +/- 0.01), and (13.84 +/- 0.66) ng/ml, respectively. The mRNA expressions of PPARgamma in the rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were significantly lower than that in control group (P < 0.05). The protein expressions of PPARgamma in the rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group were 80%, 74%, and 61% of that in control group (P < 0.05). After the cells were treated with rapamycin 100 nmol/L, PPARgamma blocking agent GW9662 10 micromol/L, and PPARgamma agonist troglitazone 10 micromol/L, respectively, for 96 hours, the mRNA expression of PPARgamma was (0.60 +/- 0.14), (0.67 +/- 0.03), and (1.30 +/- 0.14) of that in control group (P < 0.05). The protein expression showed a similar trend with mRNA expression (P < 0.05). After the cells were treated with rapamycin 100 nmol/L, PPARgamma blocking agent GW9662 10 micromol/L, and PPARgamma agonist troglitazone 10 micromol/L, respectively, for 96 hours, the expression of leptin in the control group, rapamycin 50 nmol/L group, rapamycin 100 nmol/L group, and rapamycin 200 nmol/L group was (19.02 +/- 0.52), (15.62 +/- 0.10), and (14.45 +/- 1.01) and (18.07 +/- 0.66) ng/ml, respectively (P < 0.05 compared with the control group). CONCLUSIONS: By downregulating the expression of PPARgamma, rapamycin can decrease cholesterol accumulation in 3T3-L1 cells and inhibit its leptin-secreting capability. This finding may provide a possible explanation for rapamycin-induced hyperlipidemia in clinical practice.
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Adipócitos/metabolismo , Colesterol/metabolismo , Leptina/metabolismo , PPAR gama/metabolismo , Sirolimo/farmacologia , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Animais , Camundongos , PPAR gama/genéticaRESUMO
OBJECTIVE: To explore whether overexpression of P-selectin glycoprotein ligand-1 (PSGL-1) promotes the adhesive ability of endothelial progenitor cells and functionally facilitates neovascularization in mouse model of hindlimb ischemia. METHODS: Rat endothelial progenitor cells were transfected with recombinant adenovirus vector encoding human PSGL-1. The mRNA and protein expression levels of PSGL-1 were measured by RT-PCR and Western blot, respectively. The effect of overexpression of PSGL-1 in endothelial progenitor cells was analyzed by adherence assay. Histological examination of skeletal muscle sections retrieved from the mouse ischemic hindlimbs was performed, and the hindlimb blood flow was measured by laser Doppler flow meter. RESULTS: Adenovirus vector expressing of PSGL-1 gene was successfully constructed with high titer of 3.1 × 10¹¹ pfu/ml. After transfection, PSGL-1 gene was highly expressed in the transfected endothelial progenitor cells. In vitro assay showed that overexpression of PSGL-1 enhanced the adhesive properties of endothelial progenitor cells. When the transfected endothelial progenitor cells were transplanted into the ischemic hindlimb of nude mice, the number of new capillary vessels was (41.0 ± 2.2)/HPF compared to that of (21.0 ± 2.5)/HPF in the negative control group and (10.0 ± 1.6)/HPF in the blank control group (P < 0.01). Furthermore, the blood flow was increased in the experimental group (119.1% ± 7.0%), whereas in the negative control group, it was (93.3% ± 3.0%) and in the blank control group it was (76.3% ± 12.0%), P < 0.01. CONCLUSIONS: Overexpression of PSGL-1 enhances the adhesive and angiogenic properties of endothelial progenitor cells. The approach may provide an effective therapeutic strategy to improve the efficiency of cell-based proangiogenic therapy.
Assuntos
Adesão Celular , Glicoproteínas de Membrana/biossíntese , Neovascularização Fisiológica , Células-Tronco/citologia , Adenoviridae/genética , Animais , Células Cultivadas , Células Endoteliais/citologia , Membro Posterior/irrigação sanguínea , Humanos , Isquemia/terapia , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Nus , Neovascularização Fisiológica/fisiologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Transplante de Células-Tronco , Células-Tronco/metabolismo , TransfecçãoRESUMO
P-selectin glycoprotein ligand-1 (PSGL-1) not only functions as an anchor molecule to capture monocytes and other leukocytes to endothelial cells in ischemic tissue by its interaction with P-selectin, but also transduces signals to initiate firm adhesion. Endothelial progenitor cells are derived from monocytes and play a very important role in neovascularization. Transplantation of endothelial progenitor cells is a promising therapeutic strategy to improve treatment of ischemic disease such as myocardial and cerebral infarction; however, its efficacy is now limited by the fact that few of the transplanted cells adhere to and accumulate in the ischemic tissue. In this study we aimed to investigate whether the overexpression of PSGL-1 gene promotes endothelial progenitor cells adhesion activity and explore the underlying mechanisms. We found that after transfection with human PSGL-1 gene, endothelial progenitor cells exhibited higher affinity to activated human umbilical vein endothelial cells or recombined P-selectin/ICAM-1 monolayer. The overexpression of PSGL-1-enhanced beta2-integrin expression on endothelial progenitor cells surface, and this effect was Syk dependent. The specific Syk inhibitor abolished the elevating effect of overexpression of PSGL-1 on surface beta2-integrin expression and the adhesive affinity of endothelial progenitor cells. These results suggested that Syk plays a key role in signal transduction downstream of PSGL-1 in endothelial progenitor cells, and the overexpression of PSGL-1 improves endothelial progenitor cells adhesive properties through enhanced activation of Syk and following integrin activation.