RESUMEN
A novel method for simultaneous determination of five estrogens and four androgens by online solid-phase extraction (SPE) coupled with high-performance liquid chromatography-tandem mass spectrometry (HPLC-ESI-MS/MS) in water samples was developed. An aliquot of 50 mL water sample after filtration was injected directly into autosampler and the analytes were preconcentrated on a NG1 online SPE column. After cleanup step the analytes were eluted in back flush mode and then separated on a liquid chromatography column. The experimental parameters, such as sample loading flow rate, cleanup condition and elution time, were optimized in detail. Estrogens and androgens were detected in negative and positive mode, respectively. High ionization efficiency of all the analytes was achieved by adding of 1 ammonia in the mobile phase. The recoveries ranged from 31.8% to 119.0% and the inter-day RSDs ranged from 2.7% to 19.6%. The limits of detections (LODs) were between 0.1 and 2.5 ng/L. The proposed method was successfully applied to the analysis of three types of water samples, including river water, influent and effluent water from a wastewater treatment plant (WWTP). The recoveries of androgens were not that good and a further study is being planned to improve the sensitivity for them. The proposed method is simple, sensitive and suitable for simultaneous analysis and monitoring of estrogens and androgens in water samples.
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Andrógenos/análisis , Cromatografía Líquida de Alta Presión/métodos , Estrógenos/análisis , Extracción en Fase Sólida/métodos , Contaminantes Químicos del Agua/análisis , Andrógenos/química , Andrógenos/aislamiento & purificación , Estrógenos/química , Estrógenos/aislamiento & purificación , Límite de Detección , Reproducibilidad de los Resultados , Ríos/química , Espectrometría de Masas en Tándem/métodosRESUMEN
BACKGROUND: Most patients with acute myelogenous leukemia (AML) suffer from disordered hemostasis. We have previously shown that annexin II (Ann II), a high-affinity co-receptor for plasminogen/tissue plasminogen activator, plays a central role in primary hyperfibrinolysis in patients with acute promyelocytic leukemia (APL). The expression of Ann II in cells from patients with major subtypes of AML and the effect of arsenic trioxide (As2O3) on Ann II expression in AML cells were investigated to determine whether As2O3-mediated downregulation of Ann II could restore hemostatic stability. METHODS: A total of 103 patients (48 females and 55 males; age, 19 - 58 years) were included. Plasma samples were collected before and after treatment as well as after complete remission. Ann II and plasminogen activation were measured in leukemic cells during treatment with 1 micromol/L As2O3. RESULTS: Before As2O3 treatment, Ann II mRNA expression (real-time PCR) was the highest in M3 cells (P < 0.05), higher in M5 cells than that in M1, M2, M4, and M6 cells (P < 0.001), and positively correlated with Ann II protein expression (flow cytometry) (r = 0.752, P < 0.01). Exposure for up to 120 hours to As2O3 (1 micromol/L) had no significant effect on Ann II protein in M1 and M2 leukemic cells, but decreased Ann II protein expression twofold within 48 hours of exposure in M3 cells (P < 0.05) and twofold within 96 hours in M5 cells (P < 0.05). The rate of plasmin generation was higher in APL, M5, and M4 cells than in M1, M2, and M6 cells. CONCLUSIONS: As2O3 may reduce hyperfibrinolysis in AML by downregulation of Ann II. Furthermore, As2O3 affects more than one form of AML (APL, M4 and M5), suggesting its potential role in their management.
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Anexina A2/metabolismo , Arsenicales/farmacología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Leucemia Promielocítica Aguda/metabolismo , Óxidos/farmacología , Adulto , Trióxido de Arsénico , Células de la Médula Ósea/citología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Femenino , Humanos , Leucemia Promielocítica Aguda/fisiopatología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
The aim of this study was to investigate the gene expression profiles of acute promyelocytic leukemia (APL) cell line NB4 treated with arsenic trioxide (As2O3) by using cDNA microarray. cDNA probes were prepared through reverse transcription from mRNA of NB4 cells treated with or without arsenic trioxide. The probes were labeled with Cy3 and Cy5 fluorescence dyes individually, hybridized with cDNA microarray representing 201 different human genes, and their fluorescent intensities were scanned. The genes were screened through the analysis of the difference in the gene expression profile. The results showed that after the treatment of arsenic trioxide (2 micromol/L), 6 genes were up-regulated, and 12 genes related to apoptosis and signal transduction were down-regulated. The p21, survivin, cdc2 and Wee1Hu genes may be related to the differentiation and/or apoptosis of NB4 cells induced by As2O3. It is concluded that p21, survivin, cdc2 and Wee1Hu may play an important role in the mechanism underling arsenic trioxide-mediated NB4 cell apoptosis.
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Apoptosis/genética , Arsenicales/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Leucemia Promielocítica Aguda/patología , Proteínas Asociadas a Microtúbulos/metabolismo , Óxidos/farmacología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Trióxido de Arsénico , Proteína Quinasa CDC2 , Línea Celular Tumoral , Ciclina B/metabolismo , Quinasas Ciclina-Dependientes , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Inhibidoras de la Apoptosis , SurvivinRESUMEN
OBJECTIVE: To explore the role of PTEN gene in the regulation of tissue factor (TF) expression in human neuroblastoma cells. METHODS: Expression of PTEN or TF was determined by Western blotting. Transcription of TF was examined by RT-PCR. PTEN gene expressing vector pCMV-PTEN was transfected with Lipofectamine2000. Phosphorylation of AKT was inhibited by LY294002 and then examined by Western blotting. RESULTS: Human neuroblastoma cell line SK-N-SH was PTEN-positive and expressed low level TF, whereas an other neuroblastoma cell line SK-N-MC was PTEN-negative but expressed high level TF. TF level was downregulated in SK-N-MC cells by enforced expression of PTEN in a dose dependent manner. Inhibition of TF was achieved along with inactivation of AKT. Furthermore treatment with PI3K/AKT inhibitor LY294002 also resulted in decrease of TF expression in a dose-dependent manner. CONCLUSION: Expression of TF is inhibited by PTEN gene via inactivating PI3K/AKT pathway, loss of PTEN might be the explanation of aberrant high-level TF in human neuroblastoma. It may be at least one of the mechanisms by which loss of PTEN expression confers to cancer progression.
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Neuroblastoma/metabolismo , Fosfohidrolasa PTEN/genética , Tromboplastina/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Vectores Genéticos , Humanos , Neuroblastoma/genética , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Tromboplastina/genética , TransfecciónRESUMEN
OBJECTIVE: To investigate the effects of mangiferin on telomerase activity and apoptosis in K562 cells lines and study the melocular mechanism of it's antileukemic. METHODS: Cell apoptosis was observed by light microscopy and transmission electron microscopy; The expression of Fas was measured by flow cytometry; Polymerase chain reaction enzyme linked immunoassay (PCR-ELISA) was used to assay telomerase activity of K562 cells. RESULTS: Mangiferin could inhibit telomerase activity of K562 cells in a time-and-concentration-dependent manner. Meanwhile, it could induce apoptosis obviously and up-regulate the levels of Fas in K562 cells. CONCLUSION: Mangiferin can inhibit telomerase activity of K562 cells, and the mechanism of effect is maybe relate to inducing apoptosis and the expression of Fas protein.
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Apoptosis/efectos de los fármacos , Telomerasa/metabolismo , Xantonas/farmacología , Citometría de Flujo , Humanos , Células K562RESUMEN
This study was aimed to investigate the expression and regulation mechanism of DNA-dependent protein kinase catalylic subunit (DNA-PKcs) in chronic myeloid leukemia (CML) and its role in blast crisis of CML. Expression of DNA-PKcs mRNA was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and DNA-PKcs protein by Western blot in 62 CML patients and K562, as compared to those of 23 normal individual controls. In 26 CML patients received allogeneic peripheral blood stem cell transplantation (allo-PBSCT) and 4 CML patients treated with imatinib, the expression of bcr-abl mRNA and DNA-PKcs protein was detected by RT-PCR and Western blot, respectively. After treatment with imatinib in mononuclear cell (MNC) of CML patients and K562 in vitro, expression of DNA-PKcs mRNA was detected by RT-PCR and DNA-PKcs protein level, tyrosine phosphorylation of bcr-abl fusion protein were detected by Western blot. The results showed that the expression of DNA-PKcs protein was significantly lower in CML and K562 than those in normal control (P<0.05). In 26 CML patients received allo-PBSCT and 4 CML patients treated with imatinib, the expression of DNA-PKcs protein was enhanced while the expression of bcr-abl mRNA decreased. After treatment of MNC of CML and K562 with imatinib in vitro, the expression of DNA-PKcs protein was enhanced while tyrosine phosphorylation of bcr-abl fusion protein decreased. It is concluded that the expression of DNA-PKcs protein is down-regulate by bcr-abl fusion gene, and the bcr-abl fusion gene down-regulate the expression of DNA-PKcs protein by post-transcriptional mechanism; the decrease of DNA-PKcs protein expression may be one of mechanisms underlying the acute transformation of CML.
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Proteína Quinasa Activada por ADN/genética , Proteínas de Fusión bcr-abl/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Trasplante de Células Madre de Sangre Periférica , Adulto , Anciano , Benzamidas , Células de la Médula Ósea/metabolismo , Proteína Quinasa Activada por ADN/biosíntesis , Femenino , Proteínas de Fusión bcr-abl/biosíntesis , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , Masculino , Persona de Mediana Edad , Piperazinas/uso terapéutico , Pirimidinas/uso terapéutico , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
To investigate the effect of arsenic trioxide (As(2)O(3)) or all-trans retinoic acid (ATRA) on the mRNA and protein expression of tissue factor (TF) and thrombomodulin (TM) and procoagulant activity (PCA) in NB4 cells. The NB4 cells were cultured in vitro and treated with As(2)O(3) or ATRA, expression of TF and TM antigen, and PCA change of treated NB4 cells were detected with ELISA, TF and TM mRNA transcription on the NB4 cells was assayed with reversed transcription polymerase chain reaction (RT-PCR). The results showed that 1 micromol/L As(2)O(3) and 1 micromol/L ATRA both gradually downregulated the expression of TF antigen and mRNA on NB4 cells, a human promyelocytic leukemia cell line, in time-dependent manner, as compared with control. The levels of TF antigen expression in AS(2)O(3) group were 13.3 +/- 1.8, 8.6 +/- 1.9, 10.8 +/- 1.5, 2.0 +/- 0.6 and 2.6 +/- 0.9 ng/10(7) respectively; while the levels of TF antigen expression in ATRA group were 12.4 +/- 1.1, 11.3 +/- 1.8, 5.7 +/- 1.7, 2.8 +/- 0.8 and 2.0 +/- 0.6 ng/10(7) at 24, 48, 72, 96 and 120 hours respectively (P<0.05). The procoagulant activity (PCA) of NB4 cells was decreased, blood coagulation times were 123.5 +/- 10.5, 156.3 +/- 11.6, 179.3 +/- 15.3, 248.9 +/- 20.1, 312.0 +/- 29.8 seconds in As(2)O(3) groups, respectively; 76.4 +/- 5.6, 146.8 +/- 10.9, 198.2 +/- 15.6, 265.8 +/- 20.6 and 363.8 +/- 31.9 seconds in ATRA groups respectively at 24, 48, 72, 96 and 120 hours (P<0.05). ATRA upregulated TM antigen expression on NB4 cells. It is concluded that the As(2)O(3) and ATRA decrease mRNA transcription of TF, downregulate expression of TF and reduce procoagulant activity in NB4 cells. The TM transcription and expression upregulated by ATRA may alleviate dysfunction of coagulation in APL.
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Arsenicales/farmacología , Leucemia Promielocítica Aguda/metabolismo , Óxidos/farmacología , Trombomodulina/biosíntesis , Tromboplastina/biosíntesis , Tretinoina/farmacología , Antineoplásicos/farmacología , Trióxido de Arsénico , Humanos , Leucemia Promielocítica Aguda/patología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Trombomodulina/genética , Tromboplastina/genética , Tromboplastina/metabolismo , Células Tumorales CultivadasRESUMEN
OBJECTIVE: To investigate the regulation of tissue factor (TF) on doxorubicin-induced apoptosis in human neuroblastoma. METHOD: The expression of TF was examined by Western blotting. TF siRNA-pSUPER plasmid was constructed by inserting a specific 19-nt silencing sequence targeting TF gene into pSUPER vector. Transfection of TF siRNA-pSUPER was performed using lipofectamine 2000. The activation of caspase-3 and PARP induced by doxorubicin was tested by Western blotting. The apoptotic cells were stained by Hochest 33342 and counted under fluorescence inverted microscope. RESULTS: (1) Human neuroblastoma cell line SK-N-MC expressed high level of TF. (2) Downregulation of TF expression was achieved by transfection of TF siRNA-pSUPER into SK-N-MC cells in a dose-dependent manner. (3) Cleavage of caspase-3 and PARP was increased in transfected SK-N-MC cell with down-regulation of TF. (4) TF siRNA treatment at 1 microg/ml for 8 h significantly increased apoptotic cell number in transfected SK-N-MC cells compared to that in non-transfected cells (P < 0.05) while exposing to 1 microg/ml doxorubicin for 8 h. CONCLUSIONS: Downregulation of TF expression by specific siRNA vector could increase the cytotoxicity of doxorubicin and enhance doxorubicin-induced apoptosis in human neuroblastoma cells.
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Apoptosis/efectos de los fármacos , Doxorrubicina/farmacología , Neuroblastoma/patología , Interferencia de ARN , Tromboplastina/genética , Apoptosis/genética , Caspasa 3/metabolismo , Línea Celular Tumoral , Vectores Genéticos , Humanos , Neuroblastoma/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , ARN Interferente Pequeño/genética , Tromboplastina/metabolismo , TransfecciónRESUMEN
OBJECTIVE: To investigate the expression and regulation mechanism of mismatch repair (MMR) genes in chronic myeloid leukemia (CML). METHODS: Expression of MMR genes hMSH2, hMSH3, hMSH6, hMLH1 and hPMS2 mRNAs in 62 CML patients and K562 cell line were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Expression of bcr-abl mRNA and MMR genes mRNA were detected by RT-PCR in 26 CML patients with allogeneic peripheral blood stem cell transplantation (allo-PBSCT) and 4 CML patients on imatinib treatment. Expression of bcr-abl mRNA was detected by RT-PCR and tyrosine phosphorylation of BCR-ABL fusion protein by Western blot. RESULTS: Expression of hMSH2, hMSH3 and hMLH1 mRNA was significantly lower in CML and K562 cells than in normal control (P < 0.05). In 26 CML with allo-PBSCT and 4 CML patients on imatinib treatment, expressions of hMSH2, hMSH3 and hMLH1 mRNA was enhanced while expression of bcr-abl mRNA decreased. In CML MNC after imatinib treatment and in K562 cells, expression of hMSH2, hMSH3 and hMLH1 mRNA was enhanced while tyrosine phosphorylation of BCR-ABL fusion protein decreased. CONCLUSION: Expressions of hMSH2, hMSH3 and hMLH1 mRNA were down-regulated by bcr-abl fusion gene.
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Reparación de la Incompatibilidad de ADN , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Adulto , Anciano , Antineoplásicos/farmacología , Benzamidas , Femenino , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Mesilato de Imatinib , Células K562 , Masculino , Persona de Mediana Edad , Piperazinas/farmacología , Pirimidinas/farmacología , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
To investigate the role of anti-inflammatory cytokine in acute coronary syndrome (ACS), the effect of IL-10 on expression of tissue factor (TF) induced by IL-6 in peripheral blood mononuclear cells (PBMNC) were studied. PBMNC were allowed to culture with rhIL-10 before being stimulated by rhIL-6. One-step recalcification clotting time was used to evaluate procoagulant activity (PCA) of PBMNC. The expression and activity of TF protein were determined by ELISA and cell chromogenic substrate assay. The results showed that the expression of PCA, TF protein and its activity in PBMNC increased significantly after being stimulated by rhIL-6 (P < 0.01). In PBMNC, rhIL-6-induced PCA was regulated by rhIL-10 in different doses. This effect was associated with reduction of TF protein expression and activity by rhIL-10 (P < 0.01). In conclusion, IL-10 down-regulated expression PCA and TF in PBMNC, inhibitory effect of IL-10 on expression and activity of PBMNC TF may be important protective mechanism for ACS, regulation imbalance between inflammatory and anti-inflammatory cytokines may be important factor participating in coronary thrombosis.
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Interleucina-10/farmacología , Interleucina-6/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Tromboplastina/biosíntesis , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Humanos , Interleucina-6/genética , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
OBJECTIVE: To explore the role of tissue factor/activated factor VII (TF/FVIIa) complex in human ovarian cancer invasion and metastasis. METHODS: (1) Constructed an expression vector of TF, pcDNA3-TF and established a human ovarian cell line A2780/TF expressing high level TF by using molecular cloning and gene transfection techniques. (2) By Boyden chamber assay to count the numbers of A2780 and A2780/TF cells that penetrated the matrigel to the back of PVPF membrane after FVIIa stimulation. (3) BALB/c nude mice were used to establish experimental model of metastasis with A2780 or A2780/TF and the lung tissue sections were examined by microscopy for cancer metastasis. RESULTS: (1) Compared with their parental A2780 cells, A2780/TF cells expressed high level of TF mRNA (3.99 +/- 0.15 vs 0.97 +/- 0.23, P < 0.01) and TF antigen on cell surface \[(48.56 +/- 9.53)% vs (2.73 +/- 1.15)%, P < 0.01\]. (2) After stimulation, the A2780/TF cell number on the back of PVPF membrane increased from basal level 157.3 +/- 19.2 to 447.7 +/- 39.4 (P < 0.01), which could decreased to basal level when coincubated with anti-TF antibody. (3) Cancer metastasis was found in 22.2% of nude mice transplanted with A2780 cells, while in 88.9% of those transplanted with A2780/TF cells. CONCLUSION: TF could promote the invasion and metastasis of human ovarian cancer cells through TF/FVIIa pathway.
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Factor VIIa/genética , Neoplasias Ováricas/patología , Tromboplastina/genética , Animales , Línea Celular Tumoral , Movimiento Celular , Clonación Molecular , Factor VIIa/fisiología , Femenino , Vectores Genéticos , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Metástasis de la Neoplasia , Trasplante de Neoplasias , Neoplasias Ováricas/genética , Tromboplastina/fisiología , Transfección , Trasplante HeterólogoRESUMEN
OBJECTIVE: To evaluate the efficacy and safety of human recombinant thrombopoietin (rhTPO, a product of Sunshine Pharmaceutical Co Ltd, China) in chronic refractory idiopathic thrombocytopenic purpura. METHODS: Eighty-two patients with chronic refractory idiopathic purpura received daily subcutaneous administration of rhTPO at a dose of 1.0 micro g/kg for a maximum of 14 doses. RESULTS: After the beginning of treatment, the median platelet counts increased from 15.5 (6.0 - 24.0) x 10(9)/L to 27.5 (16.0-47.0) x 10(9)/L, 35.0 (20.5-78.0) x 10(9)/L and 77.0 (41.8-119.5) x 10(9)/L on the fifth, seventh and fifteenth day, respectively (P < 0.01). After the discontinuation of rhTPO administration, the platelet count decreased gradually. On the twenty-eighth day, the median platelet count was 76.5 (35-120.3) x 10(9)/L, which was still significantly higher than the level before the treatment (P < 0.01). The overall response rate was 85.3%. The rate in the group with remarkable response (platelet count > or = 100 x 10(9)/L without bleeding) was 58.5% and that in the good response group (platelet count > or = 50 x 10(9)/L or 30 x 10(9)/L higher than the count before the treatment, without bleeding) was 26.8%. Only 3 patients had mild clinical untoward reactions. Low titer (1:5) of anti-TPO antibody in serum was detected with ELISA in one of the sixteen patients who received the test on the twenty-first and twenty-eighth day. Neutralizing test using TPO-dependent cell line showed that the positive serum had no neutralizing activity on rhTPO. CONCLUSION: Consecutive subcutaneous injection of rhTPO for a maximum of 14 days was associated with a temporary elevation in platelet counts in patients with chronic refractory ITP with tolerable adverse effects.
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Púrpura Trombocitopénica Idiopática/tratamiento farmacológico , Trombopoyetina/uso terapéutico , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Recuento de Plaquetas , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/uso terapéutico , Trombopoyetina/administración & dosificaciónRESUMEN
OBJECTIVE: To construct the expression vector of human tissue factor (TF), and investigate the influence of TF/coagulant factor VIIa (FVIIa) complex on the transcriptional expression of urokinase plasminogen activator (u-PA) and u-PA receptor (u-PAR) in human ovarian cancer. METHODS: The human TF cDNA was obtained from placenta by RT-PCR and then inserted into eukaryotic expression vector pcDNA3 to obtain the TF-pcDNA3 combinant. This combinant was transfected into human ovarian cancer cell line A2780 by lipofectamine. Stably-transfected cells A2780/TF were screened. A2780 and A2780/TF cell lines were stimulated by FVIIa respectively, and the transcriptional levels of u-PA and u-PAR were examined by RT-PCR. RESULTS: (1) The constructed product was identified as TF-pcDNA3 combinant by sequencing. (2) TF was highly expressed not only at transcriptional level in the stable-transfected A2780/TF cell (transfected cell 3.91 +/- 0.28, untransfected cell 0.97 +/- 0.23, P < 0.01), but also on the membrane of the cell surface [transfected cell (48.56 +/- 9.53)%, untransfected cell (2.73 +/- 1.15)%, P < 0.01]. (3) The u-PA and u-PAR mRNA levels in A2780 cell line did not change significantly after stimulated by FVIIa; (4) While stimulated by FVIIa, the u-PAR mRNA levels in A2780/TF cells increased significantly in both dose-dependent and time-dependent manner, while the u-PA mRNA levels did not change significantly; (5) In the A2780/TF cell line the enhanced expression of u-PAR mRNA by FVIIa was significantly inhibited by coincubated with anti-TF antibody. CONCLUSION: TF/FVIIa complex could up-regulate the transcription of u-PAR in human ovarian cancer cells so as to enhance tumor invasion and metastasis.
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Factor VIIa/metabolismo , Neoplasias Ováricas/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Tromboplastina/metabolismo , Regulación hacia Arriba , Línea Celular Tumoral , Factor VIIa/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Ováricas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Tromboplastina/genética , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
OBJECTIVE: To discuss the significance of the concentration change of plasma thrombus precursor protein (TpP) in the diagnosis of disseminated intravascular coagulation (DIC) or pre-DIC. METHODS: Enzyme linked immunosorbent assay (ELISA) was used in the detection of plasma TpP in patients with different diseases (77) and normal adults (47). At the same time, other correlated markers were detected and statistical comparison was proceeded. RESULTS: The concentration of TpP in the disease group (11.20 +/- 5.10) mg/L or the DIC (13.20 +/- 7.96) mg/L and non-DIC group (8.33 +/- 6.30) was obviously higher than that in the control group (1.29 +/- 1.02, P > 0.001). The concentration TpP in the DIC group was also obviously higher than that in the non-DIC group (P > 0.01). The rate of abnormal level of TpP in the DIC group was 100%, being higher than that of other markers related with DIC. CONCLUSION: Results of this study and pertinent literature showed that TpP detection has higher specificity and sensitivity than D-Dimer or fibrinogen (Fbg) assay in the diagnosis of DIC or pre-DIC. It provides more referable values for the diagnosis, treatment and prognosis of DIC.
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Coagulación Intravascular Diseminada/sangre , Fibrina/análisis , Adulto , Anciano , Coagulación Intravascular Diseminada/diagnóstico , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , PronósticoRESUMEN
This study was aimed to investigate coagulation factor VII level in uremic patients with chronic renal failure and to explore theirs influence factors. The plasma levels of coagulation factor VII were detected in 30 uremic patients with chronic renal failure before and after hemodialysis for 1 month, the factor VII activity (FVII:C) was determined by one-stage coagulation method, while activated factor VII (FVIIa) was measured by one-stage coagulation method using recombinant soluble tissue factor, and factor VII antigen was detected by ELISA. The results showed that: (1) The FVIIa, FVII:C and FVIIAg levels in chronic uremic patients before hemodialysis were 4.00 +/- 0.86 microg/L, (148.5 +/- 40.4)% and (99.8 +/- 21.1)% respectively, which were significantly increased, as compared with healthy controls [2.77 +/- 1.02 microg/L, (113.1 +/- 33.0)% and (73.7 +/- 18.3)% respectively, P < 0.05]. (2) After hemodialysis the FVIIa, FVII:C and FVIIAg levels in uremic patients significantly enhanced to 5.56 +/- 1.45 microg/L, (200.8 +/- 68.7)% and (124.1 +/- 19.3)% respectively (P < 0.05). (3) The abnormal increase of coagulation factor VII was positively correlated with levels of blood uria nitrogen and serum creatinine before hemodialysis but not after hemodialysis. It is concluded that the enhanced levels of coagulation factor VII in chronic uremic patients suggested abnormal activated state, herperactivity and elevated production of factor VII which correlated with renal functional injury. The abnormality of factor VII in uremia may be aggravated by hemodialysis. Coagulation factor (FVII) may be a risk factor for cardiovascular events in uremic patients who especially had been accepted long-term hemodialysis.
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Factor VII/análisis , Uremia/sangre , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Infarto del Miocardio/sangre , Infarto del Miocardio/etiología , Diálisis Renal , Factores de Riesgo , Uremia/complicaciones , Uremia/terapiaRESUMEN
OBJECTIVE: To explore the suppression effect of human cytomegalovirus (HCMV) on megakaryocytes and their precursors and study the antiviral effect of antisense phosphorothioate deoxyoligonucleotide (ASON) against HCMV. METHODS: CD34(+) cells were induced to proliferate and differentiate committedly to megakaryocytes in a semi-solid CFU-MK culture system. Cultured cells and ASON pretreated CD34(+) cells were infected by HCMV of AD169 strain. HCMV immediate early protein (IEP) DNA and mRNA and UL36 mRNA were detected by PCR and reverse transcription-polymerase chain reaction (RT-PCR). Cytotoxicity was evaluated by MTT assay. RESULTS: HCMV AD169 suppressed the proliferation of megakaryocytes significantly. Compared with the mock group, the CFU-MK yields were decreased by 21.6%, 33.8%, and 46.3%, respectively, in 3 different titers of virus infected groups (P < 0.05). The suppression was virus titer dependent. HCMV IEP DNA, HCMV IEP mRNA and UL36 mRNA were detected in the colony cells of viral infection group. Compared with the infected group by HCMV AD169, UL36Anti treatment at 0.08 micromol/L could recover the CFU-MK yields significantly (P < 0.05). In the infected MK, which was pretreated with UL36Anti at 0.08 micromol/L, HCMV UL36 mRNA was undetectable by RT-PCR. The oligonucleotide MM(1) containing a G-to-C substitution in UL36Anti was inactive at 0.08 micromol/L but active at 0.40 micromol/L. The concentration of UL36Anti necessary to significantly affect cell growth was 90.00 micromol/L. CONCLUSIONS: HCMV AD169 infection inhibits the proliferation and differentiation of megakaryocytes and their precursors. There are early transcriptions of HCMV IE and UL36 protein in infected CFU-MK. The specific ASON has a definite anti-HCMV activity.
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Antivirales/farmacología , Citomegalovirus/genética , Células Progenitoras de Megacariocitos/efectos de los fármacos , Oligonucleótidos Antisentido/farmacología , Antígenos Virales/genética , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citomegalovirus/fisiología , Sangre Fetal/citología , Interacciones Huésped-Patógeno , Humanos , Proteínas Inmediatas-Precoces/genética , Recién Nacido , Células Progenitoras de Megacariocitos/citología , Células Progenitoras de Megacariocitos/virología , Oligonucleótidos Antisentido/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND & OBJECTIVE: Human tissue factor pathway inhibitor-2 (TFPI-2) is a newly discovered serine protease inhibitor, which inhibits plasmin, trypsin, matrix metalloproteinase (MMPs), but not urokinase, tissue-type plasminogen activators and thrombin. Earlier studies have shown that the production of TFPI-2 is downregulated during the progression of various cancers. The aim of this study was to elucidate the relationship between TFPI-2 expression and ovarian tumor migration and invasion. METHODS: Human TFPI-2 expression vector pBos-Cite-neo/TFPI-2 was transfected into ovarian tumor cells line A2780. After the transfected cells were screened by G418, transfected and nontransfected cells were examined for TFPI-2 mRNA and protein by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. The number of transfected or nontransfected cells passing through membrane of Boyden chamber was counted as the basis assessing tumor cells migratory and invasive behaviors. RESULTS: Expression of mRNA and protein of TFPI-2 were confirmed in transfected cells. In invasion assay, the number of TFPI-2-expressing cells to traverse a Matrigel-coated membrane was obviously decreased compared with that of nonexpressing cells (59.3+/-6.5 versus 109.7+/-5.5, P< 0.01); while in migration assay, no significant difference was observed between transfected and nontransfected cells (114.7+/-8.6 versus 127.3+/-7.1, P >0.05). CONCLUSION: Expression of TFPI-2 may strongly inhibit the invasive ability of ovarian tumor cells in vitro, but has no effect on the migratory ability, which provides an experimental basis for treating human ovarian tumor with gene therapy.
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Glicoproteínas/fisiología , Neoplasias Ováricas/patología , Western Blotting , Línea Celular Tumoral , Movimiento Celular , Femenino , Glicoproteínas/análisis , Glicoproteínas/genética , Humanos , Invasividad Neoplásica , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The objective of this study was to explore tissue factor (TF) expression induced by TNF-alpha in cultured human umbilical vien endothelial cells (HUVEC) and its molecular mechanism. TF expression on the surface of HUVEC, TF mRNA and nuclear factor kappaB (NF-kappaB) in HUVEC were detected by flow cytometry, RT-PCR and Western blot respectively. The results showed that TNF-alpha could enhance TF expression on the surface of HUVEC, the TF expression increase was highly consistent with the increased synthesis of TF mRNA, and the increase of TF expression was lately appeared for several hours. It was also found activation of NF kappaB at the time TF mRNA increase. In conclusion, NF-kappaB could be activated promptly after HUVEC incubated with TNF-alpha, then it was bound to TF promotor to start the TF transcription, TF mRNA expression was upregulated, that leaded to the increase of TF expression on the HUVEC surface and activated the coagulation cascade.
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Células Endoteliales/metabolismo , Tromboplastina/genética , Factor de Necrosis Tumoral alfa/farmacología , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , FN-kappa B/análisis , FN-kappa B/fisiología , ARN Mensajero/análisis , Venas Umbilicales/metabolismoRESUMEN
OBJECTIVE: To investigate the inhibitory effect of NF-kappaB decoy on tissue factor (TF) expression and FVII activation in cultured human umbilical vein endothelial cells (HUVEC), and to explore new methods for prevention and treatment of coronary heart disease. METHODS: NF-kappaB decoy transfection efficiency was detected by flow cytometry, NF-kappaB decoy's mechanism was analyzed by electrophoretic mobility shift assay (EMSA), TF mRNA was detected by RT-PCR, TF antigen expression on the surface of HUVEC by flow cytometry, FVIIa level in plasma incubated with HUVEC stimulated by TNF-alpha by rsTF one stage clotting method. RESULTS: NF-kappaB decoy could be successfully transfected into HUVEC. It could compete with the endogenous kappaB cis sequence element in the regulatory regions of TF promoter to bind transcriptional factor NF-kappaB. It could also significantly inhibit the TF mRNA, TF antigen expression on the cell surface and TF function leading to activation of FVII. CONCLUSION: NF-kappaB decoy could inhibit TF gene expression and FVII activation in cultured HUVEC and might be a potential new strategy for prevention and treatment of coronary heart disease.
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Células Endoteliales/efectos de los fármacos , Factor VII/metabolismo , Oligodesoxirribonucleótidos/farmacología , Tromboplastina/biosíntesis , Células Cultivadas , Células Endoteliales/metabolismo , Citometría de Flujo , Humanos , Oligodesoxirribonucleótidos/genética , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tromboplastina/genética , Transfección , Factor de Necrosis Tumoral alfa/farmacología , Venas Umbilicales/citologíaRESUMEN
The objective of this study is to investigate the effect of vaccination with dendritic cells pulsed with survivin antigen on activation of antileukemic T cells, and inhibiting proliferation of leukemic cells. The expression of survivin on acute leukemic cells were detected by cofocal microscopy and immunoprecipitation-Western blot. DCs collected from peripheral blood mononuclear cells were pulsed with survivin purified proteins. Stimulation index (SI) and antileukemia CTL induction were analyzed with (3)H-TdR incorporation and (51)Cr releasing assay, respectively. The phenotype of T cells and DCs were identified by flow cytometry. By immunofluorescence of bone marrow and peripheral blood mononuclear cells, survivin expression was detected in 16 out of 19 AML cases (84.2%). The results showed that survivin fluorescence distribution was in cytoplasm. DCs from peripheral blood mononuclear cells were successfully induced, with typical DC morphologic characteristic. The vaccination with dendritic cells pulsed with survivin antigen dramatically stimulated the proliferation of T cells. The DCs loading survivin activated T cells with higher CD4(+) T(H) ratio as compared with DCs group, T cells activated with DCs expressed CD8 and CD56. Survivin DCs significantly inhibited the growth of leukemic cells in vitro. In conclusion, survivin antigen expressed in the cytoplasm of leukemic cells, leukemic vaccination with DCs pulsed with survivin antigen in vitro inhibited the proliferation of leukemic cells, that may be a pathway for therapy of leukemia.