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1.
Beijing Da Xue Xue Bao Yi Xue Ban ; 55(5): 781-792, 2023 Oct 18.
Artigo em Zh | MEDLINE | ID: mdl-37807730

RESUMO

OBJECTIVE: To explore the potential mechanism of resistance to axitinib in clear cell renal cell carcinoma (ccRCC), with a view to expanding the understanding of axitinib resistance, facilitating the design of more specific treatment options, and improving the treatment effectiveness and survival prognosis of patients. METHODS: By exploring the half maximum inhibitory concentration (IC50) of axitinib on ccRCC cell lines 786-O and Caki-1, cell lines resistant to axitinib were constructed by repeatedly stimulated with axitinib at this concentration for 30 cycles in vitro. Cell lines that were not treated by axitinib were sensitive cell lines. The phenotypic differences of cell proliferation and apoptosis levels between drug resistant and sensitive lines were tested. Genes that might be involved in the drug resistance process were screened from the differentially expressed genes that were co-upregulated in the two drug resistant lines by transcriptome sequencing. The expression level of the target gene in the drug resistant lines was verified by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot (WB). The expression differences of the target gene in ccRCC tumor tissues and adjacent tissues were analyzed in the Gene Expression Profiling Interactive Analysis (GEPIA) public database, and the impact of the target gene on the prognosis of ccRCC patients was analyzed in the Kaplan-Meier Plotter (K-M Plotter) database. After knocking down the target gene in the drug resistant lines using RNA interference by lentivirus vector, the phenotypic differences of the cell lines were tested again. WB was used to detect the levels of apoptosis-related proteins in the different treated cell lines to find molecular pathways that might lead to drug resistance. RESULTS: Cell lines 786-O-R and Caki-1-R resistant to axitinib were successfully constructed in vitro, and their IC50 were significantly higher than those of the sensitive cell lines (10.99 µmol/L, P < 0.01; 11.96 µmol/L, P < 0.01, respectively). Cell counting kit-8 (CCK-8) assay, colony formation, and 5-ethynyl-2 '-deoxyuridine (EdU) assay showed that compared with the sensitive lines, the proliferative ability of the resistant lines decreased, but apoptosis staining showed a significant decrease in the level of cell apoptosis of the resistant lines (P < 0.01). Although resistant to axitinib, the resistant lines had no obvious new replicated cells in the environment of 20 µmol/L axitinib. Nuclear protein 1 (NUPR1) gene was screened by transcriptome sequencing, and its RNA (P < 0.0001) and protein expression levels significantly increased in the resistant lines. Database analysis showed that NUPR1 was significantly overexpressed in ccRCC tumor tissue (P < 0.05); the ccRCC patients with higher expression ofNUPR1had a worse survival prognosis (P < 0.001). Apoptosis staining results showed that knockdown ofNUPR1inhibited the anti-apoptotic ability of the resistant lines to axitinib (786-O, P < 0.01; Caki-1, P < 0.05). WB results showed that knocking downNUPR1decreased the protein level of B-cell lymphoma-2 (BCL2), increased the protein level of BCL2-associated X protein (BAX), decreased the protein level of pro-caspase3, and increased the level of cleaved-caspase3 in the resistant lines after being treated with axitinib. CONCLUSION: ccRCC cell lines reduce apoptosis through theNUPR1 -BAX/ BCL2 -caspase3 pathway, which is involved in the process of resistance to axitinib.


Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Axitinibe/farmacologia , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Proteína X Associada a bcl-2 , Proteínas Nucleares , Linhagem Celular Tumoral , Apoptose , Proliferação de Células
2.
Beijing Da Xue Xue Bao Yi Xue Ban ; 47(4): 577-81, 2015 Aug 18.
Artigo em Zh | MEDLINE | ID: mdl-26284388

RESUMO

OBJECTIVE: To identify the functions and mechanisms of prostaglandin E2 (PGE2)-induced bone marrow-derived progenitor cells (BMPCs) maturation and its involved angiogenesis. METHODS: BMPCs harvested by flushing through the femoral and tibial bones and cultured. This population of cells was identified by immunofluorescence staining. From which, 1 µmol/L PGE2 was taken, and quantitative-PCR (Q-PCR) and Western blot were used to detect the expressions of endothelial markers and Kruppel like factor 2 (KLF2) in BMPCs. In vitro tube formation assay was performed to demonstrate the capacity of angiogenesis. Furthermore, PGE2 and its receptors EP2 and EP4 agonists were used to elucidate the regulation of PGE2 to KLF2. RESULTS: C-kit, von Willebrand factor (vWF) and vascular endothelial cadherin expressed in BMPCs. Treatment with PGE2 (1 µmol/L) significantly increased the differentiation of BMPCs. The mRNA levels of endothelial markers platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) and endothelial nitric oxide synthase (eNOS), were significantly upregulated in about 2 folds by PGE2 detected with Q-PCR assay. Matrigel tube formation assay also demonstrated PGE2 enhanced the ability of angiogenesis in BMPCs. In addition, the expression of KLF2 increased in more than 2 folds with PGE2 treatment compared with the control. Such effect of PGE2 could be blocked by EP4 blocking peptide. CONCLUSION: Promoting the differentiation of BMPCs, PGE2 reinforced their angiogenesis by binding to the receptor of EP4 in a KLF2-dependent manner.


Assuntos
Diferenciação Celular , Dinoprostona/farmacologia , Células-Tronco Hematopoéticas/citologia , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , RNA Mensageiro , Receptores de Prostaglandina E Subtipo EP2/agonistas , Receptores de Prostaglandina E Subtipo EP4/agonistas , Regulação para Cima
3.
Biomed Environ Sci ; 34(8): 616-622, 2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34474721

RESUMO

OBJECTIVE: To evaluate multidrug resistant loop-mediated isothermal amplification (MDR-LAMP) assay for the early diagnosis of multidrug-resistant tuberculosis and to compare the mutation patterns associated with the rpoB, katG, and inhA genes at the Chinese Center for Disease Control and Prevention. METHODS: MDR-LAMP assay was evaluated using 100 Mycobacterium tuberculosis ( Mtb) isolates obtained from the National Reference Laboratory for Tuberculosis in China. Phenotypic resistance to isoniazid and rifampicin and whole-genome sequencing served as reference standards. RESULTS: The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MDR-LAMP were 85.5%, 93.6%, 96.7%, and 74.4% for the detection of resistance to isoniazid and rifampicin, respectively, and 80.5%, 92.3%, 98.6%, and 41.4% for the detection of Mtb cultured from smear-positive sputum samples, respectively. When DNA sequencing was used as the reference standard, the sensitivity, specificity, PPV, and NPV of MDR-LAMP were 93.1%, 92.3%, 97.2%, and 82.8% for the detection of katG and inhA gene mutations, respectively, and 89.1%, 88.9%, 93.4%, and 81.1% for the detection of rpoB gene mutation, respectively. CONCLUSION: MDR-LAMP is a rapid and accessible assay for the laboratory identification of rifampicin and isoniazid resistance of Mtb isolates.


Assuntos
DNA Bacteriano/análise , Farmacorresistência Bacteriana Múltipla/genética , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium tuberculosis/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Antituberculosos , Proteínas de Bactérias/genética , Catalase/genética , RNA Polimerases Dirigidas por DNA/genética , Isoniazida , Mutação , Mycobacterium tuberculosis/isolamento & purificação , Oxirredutases/genética , Fenótipo , Rifampina , Sequenciamento Completo do Genoma
4.
Infect Dis Poverty ; 10(1): 59, 2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33926548

RESUMO

BACKGROUND: Information on the prevalence and resistance spectrum of nontuberculous mycobacteria (NTM) in China is mainly based on regional or local data. To estimate the proportion of NTM cases in China, a national survey of NTM pulmonary disease was carried out based on acid-fast positive sputum samples collected in 2013. METHODS: Sputum samples collected from enrolled presumptive cases in 72 nationwide tuberculosis surveillance sites from the 31 provinces in the mainland of China were cultured using L-J medium at the National tuberculosis reference laboratory (NTRL). MALDI-TOF MS identified the species of re-cultured strains, and minimal inhibitory concentrations (MICs) were determined to evaluate the drug susceptibility of NTM isolates. Data analysis used statistical software SPSS version 22.0 for Windows statistical package. RESULTS: Of 4917 mycobacterial isolates cultured, 6.4% [317/4917, 95% confidence interval (CI) 5.8%-7.2%] were confirmed as NTM, among which 7.7% (287/3709, 95% CI 6.9%-8.6%) were from the southern region. In inland and coastal China, 87.7% (95% CI 78.7%-93.2%) and 50.0% (95% CI 43.7%-56.3%) of isolates, respectively, were slow-growing mycobacteria (SGM), with the remaining rapid growing mycobacteria (RGM). A total of 29 species were detected, Mycobacterium abscessus had higher clarithromycin-inducible resistance rates than M. massiliense (65.67% vs 2.22%). M. kansasii presented lower resistance rates in linezolid and moxifloxacin than M. avium-intracellulare complex (3.23% vs 66.67%, 0 vs 47.22%) and other SGM (3.23% vs 38%, 0 vs 26%). CONCLUSIONS: More NTM pulmonary disease was observed in the south and coastal China (P < 0.01). SGM was widely distributed, and more RGM are present in southern and coastal China (P < 0.01). The antimicrobial resistance spectrum of different NTM species was significantly different and accurate species identification would be facilitated to NTM pulmonary disease treatment.


Assuntos
Antibacterianos , Micobactérias não Tuberculosas , Antibacterianos/farmacologia , China/epidemiologia , Farmacorresistência Bacteriana , Incidência
5.
Nat Commun ; 11(1): 739, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32029730

RESUMO

Clear cell renal cell carcinoma (ccRCC) is a heterogeneous disease with features that vary by ethnicity. A systematic characterization of the genomic landscape of Chinese ccRCC is lacking, and features of ccRCC associated with tumor thrombus (ccRCC-TT) remain poorly understood. Here, we applied whole-exome sequencing on 110 normal-tumor pairs and 42 normal-tumor-thrombus triples, and transcriptome sequencing on 61 tumor-normal pairs and 30 primary-thrombus pairs from 152 Chinese patients with ccRCC. Our analysis reveals that a mutational signature associated with aristolochic acid (AA) exposure is widespread in Chinese ccRCC. Tumors from patients with ccRCC-TT show a higher mutational burden and genomic instability; in addition, mutations in BAP1 and SETD2 are highly enriched in patients with ccRCC-TT. Moreover, patients with/without TT show distinct molecular characteristics. We reported the integrative genomic sequencing of Chinese ccRCC and identified the features associated with tumor thrombus, which may facilitate ccRCC diagnosis, prognosis and treatment.


Assuntos
Carcinoma de Células Renais/genética , Neoplasias Renais/genética , Trombose/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Ácidos Aristolóquicos/toxicidade , Povo Asiático/genética , Carcinoma de Células Renais/complicações , Carcinoma de Células Renais/etiologia , China , Estudos de Coortes , Feminino , Perfilação da Expressão Gênica , Estudos de Associação Genética , Instabilidade Genômica , Humanos , Neoplasias Renais/complicações , Neoplasias Renais/etiologia , Masculino , Pessoa de Meia-Idade , Mutação , Prognóstico , Trombose/complicações , Trombose/etiologia , Sequenciamento do Exoma
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