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1.
Int J Clin Oncol ; 24(2): 141-152, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30612269

RESUMO

BACKGROUND: Simultaneous detection of multiple molecular biomarkers is helpful in the prediction of treatment response and prognosis for colorectal cancer (CRC) patients. METHODS: A 22-gene panel consisting of 103 hotspot regions was utilized in the formalin-fixed paraffin-embedded (FFPE) tissue samples of 207 CRC patients, using the next-generation sequencing (NGS)-based multiplex PCR technique. Those 22 genes included AKT1, ALK, BRAF, CTNNB1, DDR2, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FGFR3, KRAS, MAP2K1, MET, NOTCH1, NRAS, PIK3CA, PTEN, SMAD4, STK11, and TP53. RESULTS: Of the 207 patients, 193 had one or more variants, with 170, 20, and 3 having one, two, and three mutated genes, respectively. Of the total 414 variants identified in this study, 384, 25, and 5 were single-nucleotide variants, deletion, and insertion. The top four frequently mutated genes were TP53, KRAS, PIK3CA, and FBXW7. There was high consistency between the results of NGS-PCR technique and routine ARMS-PCR in KRAS and BRAF mutation detection. Univariate and multivariate analyses demonstrated that advanced TNM stage, elevated serum CEA, total variants number ≥ 2, AKT1 and PTEN mutation were independent predictors of shorter DFS; poor differentiation, advanced TNM stage, total variants number ≥ 2, BRAF, CTNNB1 and NRAS mutation were independent predictors of shorter OS. CONCLUSIONS: It is feasible to detect multiple gene mutations with a 22-gene panel in FFPE CRC specimens. TNM stage and total variants number ≥ 2 were independent predictors of DFS and OS. Detection of multiple gene mutations may provide additional prognostic information to TNM stage in CRC patients.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Inclusão em Parafina , Transcriptoma , Idoso , Neoplasias Colorretais/genética , Feminino , Formaldeído , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico
2.
Sci Rep ; 7(1): 7882, 2017 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-28801584

RESUMO

To compare protein expression levels, gene mutation and survival among Right-Sided Colon Cancer (RSCC), Left-Sided Colon Cancer (LSCC) and rectal cancer patients, 57 cases of RSCC, 87 LSCC and 145 rectal cancer patients were included retrospectively. Our results demonstrated significant differences existed among RSCC, LSCC and rectal cancer regarding tumor diameter, differentiation, invasion depth and TNM stage. No significant difference was identified in expression levels of MLH1, MSH2, MSH6, PMS2, ß-Tubulin III, P53, Ki67 and TOPIIα, and gene mutation of KRAS and BRAF among three groups. Progression Free Survival (PFS) of RSCC was significantly lower than that of LRCC and rectal cancer. In univariate analyses, RSCC, preoperative chemoradiotherapy, poor differentiation, advanced TNM stage, elevated serum CEA and CA19-9 level, tumor deposit, perineural and vascular invasion were found to be predictive factors of shorter PFS. In multivariate analyses, only differentiation and TNM stages were found to be independent predictors of PFS. In conclusion, compared with LSCC and rectal cancer, RSCC has larger tumor size, poor differentiation, advanced TNM stage and shorter survival. The shorter survival in RSCC might be attributed to the advanced tumor stage caused by its inherent position feature of proximal colon rather than genetic difference.


Assuntos
Neoplasias do Colo/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Neoplasias Retais/metabolismo , Idoso , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/genética , Neoplasias do Colo/terapia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Mutação , Estadiamento de Neoplasias , Prognóstico , Intervalo Livre de Progressão , Proteoma/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Neoplasias Retais/genética , Neoplasias Retais/terapia , Estudos Retrospectivos
3.
Medicine (Baltimore) ; 96(9): e6018, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28248859

RESUMO

RATIONALE: Primary angiitis of the central nervous system (PACNS) is an inflammatory disease involving cerebrovascular and parenchymal, and solitary tumor-like mass lesion of PACNS (TLML-PACNS) is frequently misdiagnosed as neoplastic or other inflammatory diseases. However, seizure syndrome as a first manifestation of TLML-PACNS has rarely reported before. PATIENT CONCERNS: Here, we report 2 cases of seizure syndrome, which was the first sign that presented prior to the diagnosis of TLML-PACNS by brain biopsy. DIAGNOSES: A mass lesion in the white and gray matters was detected by magnetic resonance imaging. The pathology for leptomeningeal lesion biopsy observed a transmural inflammation of the artery, with T lymphocyte infiltration. Patients were diagnosed with PACNS and epileptic seizure by biopsy and electroencephalogram. INTERVENTIONS: Patients were treated with glucocorticoid pulse therapy for 3 days, and subsequently oral prednisone was continued, in combination with immunosuppressant. OUTCOMES: Luckily, both two patients were improved after treatment, and only mild cognitive impairment remained without adverse event. LESSONS: Patient with mass lesion in CNS, which is similar to tumor, presented with seizure, headache, or cerebrovascular events without any other risk factors for stroke or tumor, should be considered the feasible with the disease of TLML-PACNS.


Assuntos
Convulsões/etiologia , Vasculite do Sistema Nervoso Central/diagnóstico , Adulto , Imagem de Difusão por Ressonância Magnética , Eletroencefalografia , Humanos , Angiografia por Ressonância Magnética , Masculino , Neuroimagem , Síndrome , Tomografia Computadorizada por Raios X , Vasculite do Sistema Nervoso Central/complicações , Adulto Jovem
4.
Oncotarget ; 8(8): 12649-12663, 2017 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-28187436

RESUMO

Dishevelled (Dvl) not only links the canonical Wnt and non-canonical Wnt pathways but can also crosstalk with other pathways. As there is no systematic study to date on Dvl in rheumatoid arthritis (RA), we explored the impact of Dvl2 on proliferation and inflammatory cytokine secretion in RA fibroblast-like synoviocytes (FLSs). Expression of Dvl2 in RA synovial tissue and RA-FLSs was measured. Dvl2 was overexpressed in collagen-induced arthritis rats and human RA-FLSs,. the apoptosis and secretion of inflammatory cytokines were observed. Genetic changes and corresponding mechanisms caused by overexpressing Dvl2 in RA-FLSs were assessed. Dvl2 was found to be overexpressed in RA synovial tissue and RA-FLSs. Overexpression of Dvl2 increased apoptosis and inhibited inflammatory cytokine secretion by RA-FLSs in vivo and in vitro, and Dvl2 inhibited expression of anti-apoptotic and inflammatory genes. One possible mechanism is that Dvl2 decreases the nuclear translocation of P65 and inhibits its ability to bind to the promoters of NF-κB target genes. Our findings reveal an underappreciated role of Dvl2 in regulating inflammation and RA-FLS apoptosis and provide insight into crosstalk between the Wnt and nuclear factor-κB (NF-κB) pathways.


Assuntos
Apoptose/fisiologia , Artrite Reumatoide/metabolismo , Proteínas Desgrenhadas/metabolismo , NF-kappa B/metabolismo , Sinoviócitos/metabolismo , Animais , Artrite Reumatoide/patologia , Western Blotting , Separação Celular , Citocinas/biossíntese , Imunofluorescência , Humanos , Imunoprecipitação , Inflamação/metabolismo , Masculino , Reação em Cadeia da Polimerase , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Receptor Cross-Talk/fisiologia , Transdução de Sinais/fisiologia
5.
J Biol Chem ; 290(33): 20580-9, 2015 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-26124271

RESUMO

Prolyl hydroxylase domain proteins (PHDs) control cellular adaptation to hypoxia. PHDs are found involved in inflammatory bowel disease (IBD); however, the exact role of PHD3, a member of the PHD family, in IBD remains unknown. We show here that PHD3 plays a critical role in maintaining intestinal epithelial barrier function. We found that genetic ablation of Phd3 in intestinal epithelial cells led to spontaneous colitis in mice. Deletion of PHD3 decreases the level of tight junction protein occludin, leading to a failure of intestinal epithelial barrier function. Further studies indicate that PHD3 stabilizes occludin by preventing the interaction between the E3 ligase Itch and occludin, in a hydroxylase-independent manner. Examination of biopsy of human ulcerative colitis patients indicates that PHD3 is decreased with disease severity, indicating that PHD3 down-regulation is associated with progression of this disease. We show that PHD3 protects intestinal epithelial barrier function and reveal a hydroxylase-independent function of PHD3 in stabilizing occludin. These findings may help open avenues for developing a therapeutic strategy for IBD.


Assuntos
Mucosa Intestinal/fisiologia , Ocludina/fisiologia , Pró-Colágeno-Prolina Dioxigenase/fisiologia , Animais , Colite/genética , Colite/prevenção & controle , Deleção de Genes , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos
6.
J Biol Chem ; 290(24): 15327-36, 2015 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-25925952

RESUMO

Intestinal epithelial cells (IECs) have critical roles in maintaining homeostasis of intestinal epithelium. Endoplasmic reticulum (ER) stress is implicated in intestinal epithelium homeostasis and inflammatory bowel disease; however, it remains elusive whether IRE1α, a major sensor of ER stress, is directly involved in these processes. We demonstrate here that genetic ablation of Ire1α in IECs leads to spontaneous colitis in mice. Deletion of IRE1α in IECs results in loss of goblet cells and failure of intestinal epithelial barrier function. IRE1α deficiency induces cell apoptosis through induction of CHOP, the pro-apoptotic protein, and sensitizes cells to lipopolysaccharide, an endotoxin from bacteria. IRE1α deficiency confers upon mice higher susceptibility to chemical-induced colitis. These results suggest that IRE1α functions to maintain the intestinal epithelial homeostasis and plays an important role in defending against inflammation bowel diseases.


Assuntos
Colite/prevenção & controle , Retículo Endoplasmático/metabolismo , Endorribonucleases/fisiologia , Mucosa Intestinal/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Endorribonucleases/genética , Homeostase , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genética
7.
Oncol Lett ; 5(2): 552-558, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23420128

RESUMO

KIT autophosphorylation caused by mutation of KIT is considered to be a critical mechanism for the oncogenesis of gastrointestinal stromal tumors (GISTs). However, little is known regarding whether stem cell factor (SCF), the KIT ligand, is able to induce the proliferation of GIST cells by activating the wild-type KIT receptor in GISTs. Imatinib, a tyrosine kinase inhibitor, has been demonstrated to be effective as treatment for the majority of GISTs. However, primary resistance to imatinib in GISTs with wild-type KIT and acquired resistance in GISTs with mutant KIT are becoming increasingly significant problems. The aims of this study were to detect the expression and function of SCF in 68 GIST samples, and to explore the relationship between SCF activity and imatinib resistance using immunohistochemical staining and western blot analysis. Results showed abundant expression of SCF in GISTs and demonstrated that SCF is capable of enhancing GIST cell proliferation. Similar to its ineffectiveness in wild-type GISTs, imatinib also failed to inhibit SCF-induced KIT activation in GISTs with mutant KIT. We also found increased SCF expression in GIST cells treated with imatinib. Overall, our results indicated that SCF-induced KIT activation is a novel essential pathway for the proliferation of GISTs. Imatinib was not able to inhibit the activity of SCF, while it promoted the expression of SCF, which may have contributed to acquired imatinib resistance.

8.
Chin Med J (Engl) ; 125(9): 1677-80, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22800846

RESUMO

Natural killer T-cell lymphoma (NKTL) is a malignant neoplasm which usually involves the nasal cavity or paranasal sinuses, while an orbit origin is extremely rare. Here we report the clinical, radiological and histopathologic features of a patient with NKTL originating from the orbit. We analyzed the clinical and radiologic records in the whole course of the disease. We also reviewed the morphology and immunohistochemistry of the neoplasm biopsy, including the presence of CD56, CD3 and cytotoxic molecules. This case demonstrated that nasal-type NKTL with a poor prognosis can originate from the orbit.


Assuntos
Linfoma de Células T/etiologia , Células T Matadoras Naturais/patologia , Neoplasias Orbitárias/complicações , Humanos , Linfoma de Células T/metabolismo , Masculino , Pessoa de Meia-Idade , Células T Matadoras Naturais/metabolismo , Neoplasias Orbitárias/metabolismo
9.
World J Gastroenterol ; 18(23): 2929-37, 2012 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-22736916

RESUMO

AIM: To clarify the biological role of stem cell factor (SCF)-mediated wild-type KIT receptor activation in gastrointestinal stromal tumor (GIST) growth. METHODS: The co-expression of wild-type KIT receptor and SCF was evaluated in 51 GIST samples using mutation analysis and immunohistochemistry, and the results were correlated with clinicopathological parameters, including the mitotic count, proliferative index (Ki-67 immunohistochemical staining), mitotic index (phospho-histone H3 immunohistochemical staining) and apoptotic index (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling). Using primary cultured GIST cells, the effect of SCF-mediated wild-type KIT receptor activation was determined by western blotting, methyl thiazolyl tetrazolium (MTT), and apoptosis assays. RESULTS: We found that wild-type KIT receptor and SCF protein were expressed in 100% and 76.5% of the 51 GIST samples, respectively, and the co-expression of wild-type KIT receptor and SCF was associated with known indicators of poor prognosis, including larger tumor size (P = 0.0118), higher mitotic count (P = 0.0058), higher proliferative index (P = 0.0012), higher mitotic index (P = 0.0282), lower apoptosis index (P = 0.0484), and increased National Institutes of Health risk level (P = 0.0012). We also found that the introduction of exogenous SCF potently increased KIT kinase activity, stimulated cell proliferation (P < 0.01) and inhibited apoptosis (P < 0.01) induced by serum starvation, while a KIT immunoblocking antibody suppressed proliferation (P = 0.01) and promoted apoptosis (P < 0.01) in cultured GIST cells. CONCLUSION: SCF-mediated wild-type KIT receptor activation plays an important role in GIST cell growth. The inhibition of SCF-mediated wild-type KIT receptor activation may prove to be particularly important for GIST therapy.


Assuntos
Neoplasias Gastrointestinais/metabolismo , Tumores do Estroma Gastrointestinal/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Fator de Células-Tronco/metabolismo , Adulto , Idoso , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/patologia , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/patologia , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Índice Mitótico , Mutação , Proteínas Proto-Oncogênicas c-kit/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-kit/genética , Fator de Células-Tronco/farmacologia , Células Tumorais Cultivadas
12.
Int J Colorectal Dis ; 27(4): 429-35, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22076611

RESUMO

PURPOSE: There is a lack of study concerning expression of Topoisomerase IIα (Topo IIα) and long-term results in colorectal cancer patients. We aimed to investigate the relationship between expression of Topo IIα and clinicopathological parameters including overall survival in colorectal cancer. METHODS: Paraffin-fixed specimens from a large prospective cohort of colorectal cancer patients who had been followed up for 4 years were assayed immunohistochemically. RESULTS: Of 490 colorectal cancer patients accessible for Topo IIα expression, expression of Topo IIα was scored as (-) in 4 (0.8%) patients, (+) in 41 (8.4%) patients, (++) in 396 (80.8%) patients, and (+++) in 49 (10.0%) patients. Overexpression of Topo IIα was found to be related with lower T stage (p = 0.042), lower N stage (p = 0.038), and a lower incidence of recurrence with nearly significance (p = 0.053). Kaplan-Meier analyses showed that overexpression of Topo IIα was related with prolonged overall survival (p = 0.022) and disease-free survival (p = 0.036). Multivariate analyses showed that elevated serum CEA (p < 0.001), elevated serum CA199 (p = 0.002), poor differentiation (p = 0.001), advanced Dukes stage (p < 0.001), and lower expression of Topo IIα (p = 0.017) were independent predictive factors for poor prognosis. CONCLUSIONS: Topo IIα expression is a valuable prognostic indicator for colorectal cancer and would be useful in treatment selection for early colorectal cancer and malignant colorectal polyps resected under endoscopy, especially when it is used in combination with serum CEA, CA199, and differentiation.


Assuntos
Antígenos de Neoplasias/metabolismo , Neoplasias Colorretais/enzimologia , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Idoso , Neoplasias Colorretais/patologia , Intervalo Livre de Doença , Feminino , Humanos , Mucosa Intestinal/enzimologia , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Modelos de Riscos Proporcionais
14.
Zhonghua Zhong Liu Za Zhi ; 31(7): 500-4, 2009 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-19950696

RESUMO

OBJECTIVE: To explore the effect of malignant transformation of the L839P, a new mutation site of the PDGFRA gene, on the pathogenesis of gastrointestinal stromal tumors. METHODS: All recombinant plasmids were stably transfected into CHO cells by liposomes. Western blotting was used to detect the expression of PDGFRA protein. The cell growth curve was plotted by cell counting. Flow cytometry was used to detect the cell cycle and apoptosis of CHO cell, respectively. The stably transformed cells were inoculated subcutaneously into the back of nude mice and the mice were used to observe the tumorigenesis. Transient transfection of the mutant-type plasmids of PDGFRA gene and the wild-type plasmids of kit gene into the CHO cells was performed. Western blot was used to detect the expression of kit protein and its phosphorylated forms. RESULTS: PDGFRA protein expressed in the negative control, experimental group and positive control, except the empty vector. The growth curve showed that it was accelerated in the experimental group and positive control. The ratios of cells in proliferative phase were 28.4% (blank), 24.5% (negative control), 43.8% (experimental group) and 40.9% (positive control). Their apoptotic indexes were 1.8%, 1.9%, 1.5% and 1.6%, respectively. After three weeks, tumors were observed in the nude mice of experimental group and positive control, inoculated with the stably transformed cells. Moreover, the expression of phosphorylated protein of kit was enhanced after cotransfection of the mutant-type plasmids of PDGFRA and the wild-type plasmid of kit. CONCLUSION: The PDGFRA mutant L839P is a gain-of-function mutation and has obviously malignant transforming effect on normal cells, and may activate kit protein accelerating the tumorigenesis. Gastrointestinal stromal tumors;


Assuntos
Transformação Celular Neoplásica , Tumores do Estroma Gastrointestinal/genética , Mutação , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Animais , Apoptose , Células CHO , Ciclo Celular , Proliferação de Células , Cricetinae , Cricetulus , Tumores do Estroma Gastrointestinal/etiologia , Tumores do Estroma Gastrointestinal/patologia , Camundongos , Camundongos Nus , Plasmídeos , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transfecção
15.
BMC Cancer ; 9: 9, 2009 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-19134212

RESUMO

BACKGROUND: p75NTR has been used to isolate esophageal and corneal epithelial stem cells. In the present study, we investigated the expression of p75NTR in esophageal squamous cell carcinoma (ESCC) and explored the biological properties of p75NTR+ cells. METHODS: p75NTR expression in ESCC was assessed by immunohistochemistry. p75NTR+ and p75NTR- cells of 4 ESCC cell lines were separated by fluorescence-activated cell sorting. Differentially expressed genes between p75NTR+ and p75NTR- cells were determined by real-time quantitative reverse transcription-PCR. Sphere formation assay, DDP sensitivity assay, 64copper accumulation assay and tumorigenicity analysis were performed to determine the capacity of self-renewal, chemotherapy resistance and tumorigenicity of p75NTR+ cells. RESULTS: In ESCC specimens, p75NTR was found mainly confined to immature cells and absent in cells undergoing terminal differentiation. The percentage of p75NTR+ cells was 1.6%-3.7% in Eca109 and 3 newly established ESCC cell lines. The expression of Bmi-1, which is associated with self-renewal of stem cells, was significantly higher in p75NTR+ cells. p63, a marker identified in keratinocyte stem cells, was confined mainly to p75NTR+ cells. The expression of CTR1, which is associated with cisplatin (DDP)-resistance, was significantly decreased in p75NTR+ cells. Expression levels of differentiation markers, such as involucrin, cytokeratin 13, beta1-integrin and beta4-integrin, were lower in p75NTR+ cells. In addition, p75NTR+ cells generated both p75NTR+ and p75NTR- cells, and formed nonadherent spherical clusters in serum-free medium supplemented with growth factors. Furthermore, p75NTR+ cells were found to be more resistant to DDP and exhibited lower 64copper accumulation than p75NTR- cells. CONCLUSION: Our results demonstrated that p75NTR+ cells possess some characteristics of CSCs, namely, self-renewal and chemotherapy resistance. Chemotherapy resistance of p75NTR+ cells may probably be attributable to decreased expression of CTR1.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Neoplasias Esofágicas/metabolismo , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Animais , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Processos de Crescimento Celular , Linhagem Celular Tumoral , Proliferação de Células , Cisplatino/farmacologia , Radioisótopos de Cobre/metabolismo , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/patologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Proteínas Nucleares/metabolismo , Complexo Repressor Polycomb 1 , Proteínas Proto-Oncogênicas/metabolismo , Distribuição Aleatória , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia
16.
Heart Vessels ; 23(3): 193-200, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18484163

RESUMO

Hypoxia-inducible factor (HIF)-1alpha and-2alpha have diverse actions on the myocardium, but the importance of direct effects on cardiac myocytes is unclear. To define their regional accumulation and association with cardiomyocyte cell cycle change after myocardial infarction (MI), a rat MI model was established by occluding the coronary arteries. To further prove a causative relationship between HIF and cell cycle regulation, cultured cardiomyocytes were transfected with adenoviral vectors carrying HIF-1alpha and HIF-2alpha. Two weeks after MI, both HIF-1alpha and HIF-2alpha mRNA were moderately increased in the infarcted left ventricle and noninfarcted left ventricle; HIF-2alpha amplification was also detected in areas of the interventricular septum and the right ventricle. In concordance with the changes in mRNA levels, immunohistochemistry signals of HIF-1alpha and HIF-2alpha were characterized by different regional distributions. In the myocardium adjacent to the infarcted tissue, a significant correlation between HIF-1alpha or HIF-2alpha and Ki-67 labeling index was observed (P < 0.001). Immunohistochemical double staining showed that HIF positive cardiomyocytes underwent DNA synthesis. Cardiomyocytes treated with HIF-1alpha or -2alpha expressed Ki-67, phosphohistone H3, and bromodeoxyuridine effectively in vitro. In conclusion, HIF-1alpha and HIF-2alpha had a distinct spatial expression pattern in a rat model of ischemic heart disease. Both HIF subunits might be potent stimuli for cardiomyocytes to re-enter the cell cycle and initiate DNA synthesis.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ciclo Celular , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Adenoviridae/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Bromodesoxiuridina/metabolismo , Ciclo Celular/genética , Proliferação de Células , Células Cultivadas , Replicação do DNA , Modelos Animais de Doenças , Vetores Genéticos , Histonas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Antígeno Ki-67/metabolismo , Masculino , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Miócitos Cardíacos/patologia , Fosforilação , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos Lew , Regeneração , Transfecção , Regulação para Cima
17.
Microvasc Res ; 76(1): 23-30, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18462761

RESUMO

BACKGROUND: To examine the effects of angiogenin modified mesenchymal stem cells (MSCs) on ventricular remodeling and cardiac function in a rat model of acute myocardial infarction. METHODS: MSCs were transfected with adenoviral vectors carrying either angiogenin (MSC(AdANG)) or EGFP (MSC(AdEGFP)). Angiogenin gene amplification, protein expression and cell death were assayed after hypoxic treatment. DiI labeled MSC(AdANG) and MSC(AdEGFP) were injected into infracted heart. Six weeks after cell transplantation, echocardiography and histological study were performed. RESULTS: After hypoxia treatment, angiogenin modified MSCs effectively expressed angiogenin for at least 14 days. The death of MSC(AdANG) was one-third of MSCAd(EGFP), and 50% of untreated MSCs. In the infracted myocardium, the number of DiI labeling cells in MSC(AdANG) group with high angiogenin expression was three-fold that in MSC(AdEGFP) group. Echocardiograms suggested that angiogenin modified MSCs significantly improved left ventricular (LV) systolic and diastolic function. Histological study confirmed that ventricular remodeling was attenuated significantly in MSC(AdANG) group. Furthermore, vasculogenesis was enhanced significantly in MSC(AdANG) group as measured by both factor VIII and alpha-SMA staining. CONCLUSION: Angiogenin modified MSCs enhanced the tolerance of engrafted MSCs to hypoxia injury in vitro and improved their viability in infracted hearts, thus helping preserve the LV contractile function and attenuate LV remodeling through vasculogenesis.


Assuntos
Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Infarto do Miocárdio/terapia , Neovascularização Fisiológica , Ribonuclease Pancreático/fisiologia , Actinas/análise , Adenoviridae/genética , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/metabolismo , Proteínas Angiogênicas/fisiologia , Animais , Antígenos CD/análise , Capilares/anatomia & histologia , Hipóxia Celular , Sobrevivência Celular/fisiologia , Colágeno/metabolismo , Expressão Gênica , Vetores Genéticos/genética , Antígenos HLA-DR/análise , Masculino , Células-Tronco Mesenquimais/citologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Miocárdio/metabolismo , Miocárdio/patologia , Ratos , Ratos Endogâmicos Lew , Ribonuclease Pancreático/genética , Ribonuclease Pancreático/metabolismo , Transfecção , Função Ventricular Esquerda/fisiologia , Remodelação Ventricular/fisiologia , Vimentina/análise
18.
World J Gastroenterol ; 14(10): 1575-81, 2008 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-18330951

RESUMO

AIM: To determine the inhibitory effect of the adenovirus-based angiopoietin-1 (Ang-1) targeted small interfering RNA expression system (Ad/Ang-1si) on the expression of the Ang-1 gene, cell growth and apoptosis in human esophageal cancer cell line Eca109. METHODS: siRNA-expressing adenovirus targeting Ang-1 gene was constructed using the Ad Easy System. Cultured Eca109 cells were transfected with Ad/Ang-1si (Eca109/Ang-1si), and Ad/si was used to infect Eca109 cells as control (Eca109/si). Ang-1 gene expression and concentration was determined with RT-PCR and ELISA, respectively. Human umbilical vein endothelial cell (HUVEC) migration and proliferation were analyzed. After s.c. injection into athymic nu/nu mice, the tumor growth, vessel density and apoptosis of each group was also determined. RESULTS: HUVEC migration induced by conditioned medium from Ang-1si-transfected Eca109 cells was significantly less than that induced by conditioned medium from Eca109 cells and control adenovirus-transfected Eca109 cells. Furthermore, after s.c. injection into athymic nu/nu mice, the tumor growth and cell apoptosis of Ad/Ang-1si -expressing Eca109 cells was significantly lower than that of parental or control adenovirus-transfected cells. Vessel density assessed by CD31 immunohistochemical analysis and Ang-1 expression by RT-PCR were also decreased. CONCLUSION: The targeting Ang-1 may provide a therapeutic option for esophageal cancer.


Assuntos
Angiopoietina-1/genética , Neoplasias Esofágicas/irrigação sanguínea , Neoplasias Esofágicas/patologia , Neovascularização Patológica/genética , Interferência de RNA/fisiologia , Angiopoietina-1/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/terapia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Transfecção , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Heart Vessels ; 22(2): 116-22, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17390207

RESUMO

Stable cell-matrix adhesion, re-endothelialization, and reconstitution represent important issues in creating autologous living heart valve, a close collaboration between growth factors and the extracellular matrix in these processes appears crucial. To prove this action, porcine decellularized valve constructs were precoated with fibronectin and seeded with hepatocyte growth factor-transferred marrow stromal cells (MSCs) and grown in vitro in a pulsatile-flow bioreactor. Results showed hepatocyte growth factor stimulated adhesion of MSCs to fibronectin in a time-dependent manner with a range of 8-128 ng/ml. Histological observation demonstrated a time course of MSC growth on decellularized valve constructs. A handful of cells, a loose cellular layer, a confluent monolayer coverage, a 2-layer structure and a 3-layer structure were observed at weeks 2, 3, 4, 6, and 8, respectively. Immunohistochemical analysis revealed cellular reconstitution of endothelial cells (von Willebrand factor positive) and myofibroblasts (alpha-smooth muscle actin and vimentin double-positive) at week 8. Importantly, endothelial cell retention (17.3 +/- 2.6/mm) remained high under exposure to high flow and pressure conditions in a bioreactor. These results demonstrated that the combination of fibronectin and hepatocyte growth factor contributed to creating autologous living heart valve.


Assuntos
Bioprótese , Junções Célula-Matriz/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Fibronectinas/farmacologia , Próteses Valvulares Cardíacas , Fator de Crescimento de Hepatócito/farmacologia , Células Estromais/efeitos dos fármacos , Engenharia Tecidual , Adenoviridae/genética , Animais , Células da Medula Óssea/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Vetores Genéticos , Humanos , Suínos
20.
World J Gastroenterol ; 11(25): 3948-52, 2005 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-15991300

RESUMO

AIM: To examine the prevalence and prognostic significance of C-kit gene mutation and analysis the correlation of C-kit gene mutation and the clinicalpathologic parameters of GISTs. METHODS: Eighty-two GISTs were studied for the mutation of C-kit gene by PCR-SSCP, DNA sequence. Statistical comparison were used to analysis the correlation of C-kit gene mutation and clinicalpathology, clinical behavior, recurrence. RESULTS: (1) Mutation-positive and mutation-negative GISTs were 34 and 48,respectively; (2) Among these patients with C-kit mutation remained a significantly poor prognosis associated with 59% 3-year survival compared to those whose tumors did not; (3) Tumor size, PCNA index, mitotic cell number, presence of necrosis, microscopic invasion to adjacent tissues, recurrence and distant metastasis among mutation-positive and mutation-negative GISTs were significantly different. CONCLUSION: C-kit mutation is a undoubtedly pivotal event in GIST and may be associated with poor prognosis. Evaluation of C-kit gene mutation may have both prognosis and therapeutic significances.


Assuntos
Tumores do Estroma Gastrointestinal/genética , Mutação , Proteínas Proto-Oncogênicas c-kit/genética , Adulto , Feminino , Frequência do Gene , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico
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