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1.
Cancer Genet Cytogenet ; 178(1): 1-10, 2007 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-17889702

RESUMO

Patients with multiple myeloma (MM) have increased bone marrow angiogenesis, but the angiogenic properties of myeloma cells and the mechanism of MM-induced angiogenesis have not been completely clarified. The brain-derived neurotrophic factor (BDNF) and its high-affinity receptor, TrkB, have been identified as critical factors in the regulation of vessel formation. In this study, we demonstrate that patients with MM had increased BDNF and vascular endothelial growth factor (VEGF) in their peripheral blood. We also found in particular that a decreased BDNF level was correlated with the remission of MM. BDNF was expressed by the human myeloma cell line RPMI8226 and primary myeloma cells, and TrkB was expressed by human umbilical vein endothelial cells (HUVEC) at the protein levels. In a coculture system, we observed that both RPMI8226 cells and primary myeloma cells induced the migration and formation of a net-like structure in HUVEC. The anti-BDNF monoclonal antibody significantly but partially restrained the angiogenesis effect of MM cells. Moreover, in an experimental model of angiogenesis in vivo, BDNF and VEGF significantly promoted vessel formation in Matrigel plug compared to the control. These effects were also blocked by anti-BDNF monoclonal antibody. Finally, our in vitro results were supported by the in vivo finding in human myeloma xenograft NOD/SCID models. Anti-BDNF mAb treatment resulted in inhibition of tumor growth, decreased vessel density, and tumor necrosis. Our study suggested that the BDNF/TrkB signaling pathway could be involved, at least in part, in MM-induced angiogenesis.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Mieloma Múltiplo/patologia , Neovascularização Patológica , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Pessoa de Meia-Idade , Modelos Biológicos , Transplante de Neoplasias , Receptor trkB/metabolismo
2.
Cancer Genet Cytogenet ; 169(1): 12-20, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16875931

RESUMO

The evolution of multiple myeloma (MM) depends on complex signals from the bone marrow microenvironment, which support proliferation and survival of malignant plasma cells. Previous study defined a brain-derived neurotrophic factor-tyrosine kinase receptor B (BDNF/TrkB) axis in myeloma and autocrine growth stimulation by BDNF in various tumor cells. We examined the biological effects of BDNF on MM cells. Using a reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry, we observed that both BDNF and its high-affinity receptor TrkB are expressed by MM cell lines (RPMI 8226, U266, and KM3) and primary MM cells. Functional studies revealed that BDNF was a potent growth factor for MM. BDNF (5-500 ng/mL) had strong proliferative effects on both MM cell lines and primary MM cells, shown by [(3)H]thymidine incorporation assay. BDNF (12.5-200 ng/mL) also induced migration of MM cells, as indicated by the Transwell migration assay. Together, our data indicate that BDNF is a potent myeloma growth and chemotactic factor and suggest that the BDNF/TrkB pathway is a potential therapeutic target in MM.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Divisão Celular/efeitos dos fármacos , Mieloma Múltiplo/patologia , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
Zhonghua Nei Ke Za Zhi ; 44(6): 418-20, 2005 Jun.
Artigo em Zh | MEDLINE | ID: mdl-16008850

RESUMO

OBJECTIVE: To study the variation and significance of plasma factor VII (FVII) in various types of ischemia heart disease (IHD) and discuss its relation with plasma lipid. METHODS: FVIIa was determined with one stage clotting assay using a recombinant soluble tissue factor (rsTF). FVIIc was measured with one stage clotting assay. FVIIag was quantified with enzyme-linked immunosorbent assay (ELISA). RESULTS: FVIIa in stable angina (SA), unstable angina (UA), old and acute myocardial infraction (OMI, AMI) patients were higher than that of a normal group with significant statistic differences between any two groups. FVIIag in UA, OMI and AMI were higher than that in SA and the normal group. There were positive correlations between FVIIa and serum triglycerides, FVIIa and FVIIc, FVIIc and FVIIag. CONCLUSIONS: There is more or less activation of extrinsic coagulation pathway in any kind of IHD. FVIIa is the risk factor in the development of IHD and more sensitive than FVIIc or FVIIag. FVIIa is higher in OMI which may be one of the risk factors of reinfarction. Serum triglyceride may indirectly lead to the pathologic process of IHD by increasing the level of FVIIa.


Assuntos
Fator VII/análise , Lipídeos/sangue , Isquemia Miocárdica/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Isquemia Miocárdica/fisiopatologia , Fatores de Risco
4.
Zhonghua Nei Ke Za Zhi ; 43(1): 49-51, 2004 Jan.
Artigo em Zh | MEDLINE | ID: mdl-14990023

RESUMO

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.


Assuntos
Coagulação Intravascular Disseminada/sangue , Fibrina/análise , Adulto , Idoso , Coagulação Intravascular Disseminada/diagnóstico , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico
5.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 16(1): 181-4, 2008 Feb.
Artigo em Zh | MEDLINE | ID: mdl-18315926

RESUMO

This study was aimed at clarification of the function of EGF(1) segment in rat coagulation factor VII with tissue factor (TF) by means of the expression of the fusion protein of EGFP-EGF(1). The DNA fragment encoding EGF(1) was amplified from a rat liver tissue by RT-PCR, and then inserted in an EGFP-procaryotic expression vector to construct the recombinant plasmid pET28a-EGFP-EGF(1) which was introduced into the competent cells of E.coli BL21, then an engineering bacteria strain was obtained which was induced by IPTG to express the fusion protein of EGFP-EGF(1). The fusion protein was purified by chromatography on Ni column, and then acted on the rat hemangioendotheliocytes stimulated with LPS to express TF; the binding of the fusion protein to the hemangioendotheliocytes was detected by means of fluorescence microscopy and flow cytometry. The results indicated that EGFP-EGF(1) was highly expressed in the engineering E.coli strain, and successfully purified, and its molecular mass was confirmed as 36 kD by SDS-PAGE. Fluorescence microscopy and flow cytometry had shown that this fusion protein can bind with the TF on the hemangioendotheliocytes. It is concluded that the EGF(1) region of rat coagulation factor can mediate the specific binding of FVII with TF, so as to lay partly the basis for molecular targeting anti-thrombotic therapy.


Assuntos
Células Endoteliais/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Fator VII/genética , Proteínas de Fluorescência Verde/genética , Tromboplastina/metabolismo , Animais , Fator de Crescimento Epidérmico/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Fator VII/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo
6.
Zhonghua Xue Ye Xue Za Zhi ; 28(6): 363-6, 2007 Jun.
Artigo em Zh | MEDLINE | ID: mdl-17939398

RESUMO

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.


Assuntos
Neuroblastoma/metabolismo , PTEN Fosfo-Hidrolase/genética , Tromboplastina/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos , Humanos , Neuroblastoma/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tromboplastina/genética , Transfecção
7.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 15(6): 1191-5, 2007 Dec.
Artigo em Zh | MEDLINE | ID: mdl-18088464

RESUMO

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.


Assuntos
Apoptose/genética , Arsenicais/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Leucemia Promielocítica Aguda/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Óxidos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Trióxido de Arsênio , Proteína Quinase CDC2 , Linhagem Celular Tumoral , Ciclina B/metabolismo , Quinases Ciclina-Dependentes , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Inibidoras de Apoptose , Survivina
8.
Zhonghua Xue Ye Xue Za Zhi ; 28(9): 594-7, 2007 Sep.
Artigo em Zh | MEDLINE | ID: mdl-18246814

RESUMO

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.


Assuntos
Apoptose/efeitos dos fármacos , Doxorrubicina/farmacologia , Neuroblastoma/patologia , Interferência de RNA , Tromboplastina/genética , Apoptose/genética , Caspase 3/metabolismo , Linhagem Celular Tumoral , Vetores Genéticos , Humanos , Neuroblastoma/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno/genética , Tromboplastina/metabolismo , Transfecção
9.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 15(2): 391-5, 2007 Apr.
Artigo em Zh | MEDLINE | ID: mdl-17493354

RESUMO

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.


Assuntos
Arsenicais/farmacologia , Leucemia Promielocítica Aguda/metabolismo , Óxidos/farmacologia , Trombomodulina/biossíntese , Tromboplastina/biossíntese , Tretinoína/farmacologia , Antineoplásicos/farmacologia , Trióxido de Arsênio , Humanos , Leucemia Promielocítica Aguda/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Trombomodulina/genética , Tromboplastina/genética , Tromboplastina/metabolismo , Células Tumorais Cultivadas
10.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 13(3): 479-82, 2005 Jun.
Artigo em Zh | MEDLINE | ID: mdl-15972146

RESUMO

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.


Assuntos
Interleucina-10/farmacologia , Interleucina-6/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Tromboplastina/biossíntese , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Humanos , Interleucina-6/genética , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Proteínas Recombinantes/farmacologia
11.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 13(1): 104-9, 2005 Feb.
Artigo em Zh | MEDLINE | ID: mdl-15748446

RESUMO

In order to investigate the expression of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) in multiple myeloma patients and the in vitro and in vivo proangiogenic effects of BDNF, the plasma concentrations of BDNF and VEGF in MM patients and control group were determined by ELISA, the effect of BDNF on the in vitro proliferation of human umbilical vein endothelial cells (HUVEC) was examined by MTT assay; the effects of BDNF on HUVEC migration and tube formation were studied by modified Boyden chamber assay and tube formation assay, respectively. Matrigel plug assay and chorioallantoic membrane assay were used to evaluate the effect of BDNF on angiogenesis in vivo. The results demonstrated that the concentration of BDNF was (4.22 +/- 0.64) ng/ml and (2.03 +/- 0.38) ng/ml in MM group and control group, respectively, (P = 0.01). There was also a significant difference between VEGF levels of two groups [(79.35 +/- 13.25) pg/ml vs (34.41 +/- 1.78) pg/ml, P = 0.006]. The levels of BDNF and VEGF correlated significantly (r = 0.430, P = 0.025). BDNF stimulated the migration and tube formation in vitro significantly, although it had no effect on the proliferation of HUVEC. BDNF also stimulated angiogenesis both in matrigel plug of mouse model and in chick chorioallantoic membrane. It is concluded that the concentrations of BDNF and VEGF in MM patients' peripheral blood are at high level; BDNF can stimulate the angiogenesis markedly in vitro and in vivo. Therefore, BDNF may act as an important regulator in angiogenesis of MM.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/sangue , Mieloma Múltiplo/sangue , Fator A de Crescimento do Endotélio Vascular/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Indutores da Angiogênese/sangue , Indutores da Angiogênese/farmacologia , Animais , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Membrana Corioalantoide/embriologia , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/farmacologia
12.
Zhonghua Xue Ye Xue Za Zhi ; 26(9): 534-8, 2005 Sep.
Artigo em Zh | MEDLINE | ID: mdl-16468330

RESUMO

OBJECTIVE: To investigate a new strategy of polylactic acid (PLA) nanoparticles delivery system coating nuclear factor-kappaB (NF-kappaB) decoy oligonucleotides (ODNs) for inhibiting TF expression in cultured brain microvascular endothelial cells(BMECs). METHODS: PLA nanoparticles coating FITC-labeled NF-kappaB decoy ODNs were formulated by nano-deposition method and the characteristics of nanoparticles were detected. BMECs were isolated and cultured in vitro. The cellular uptake and intracellular localization of nanoparticles in BMECs was detected by flow cytometry and confocal microscopy. Changes in the expressions of TF and nuclear protein P65 were examined by RT-PCR and Western blot in NF-kappaB decoy ODNs transfected BMECs by LPS stimulation. RESULTS: The decoy-nanoparticles obtained were uniform spherical particles with an effective diameter of 162.1 nm and a polydispersity index of 0.118. NF-kappaB decoy ODNs encapsulated in nanoparticles could be released in a controlled manner in phosphate-buffered saline for up to 28 days. It was observed that the cellular uptake of nanoparticles were increased with the time of incubation and the concentration of nanoparticles in the medium. Nanoparticles localized mainly in the BMECs cytoplasm. LPS-induced upregulation of TF transcription was inhibited by NF-kappaB decoy ODNs transfection but not by missense ODNs transfection. Furthermore, changes in the transcription level of TF were paralleled by a reduction of capacity of P65 in nuclear extract of NF-kappaB decoy ODNs transfected cells. CONCLUSIONS: These findings offer a potential therapeutic strategy in the control of TF expression in BMECs in vitro.


Assuntos
Células Endoteliais/metabolismo , NF-kappa B/genética , Oligonucleotídeos/genética , Tromboplastina/metabolismo , Animais , Encéfalo/irrigação sanguínea , Capilares/citologia , Células Cultivadas , Regulação da Expressão Gênica , Ácido Láctico , Nanopartículas , Poliésteres , Polímeros , Ratos , Tromboplastina/genética , Transfecção
13.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 11(2): 124-7, 2003 Apr.
Artigo em Zh | MEDLINE | ID: mdl-12744731

RESUMO

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.


Assuntos
Células Endoteliais/metabolismo , Tromboplastina/genética , Fator de Necrose Tumoral alfa/farmacologia , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , NF-kappa B/análise , NF-kappa B/fisiologia , RNA Mensageiro/análise , Veias Umbilicais/metabolismo
14.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 11(6): 579-82, 2003 Dec.
Artigo em Zh | MEDLINE | ID: mdl-14706138

RESUMO

The aim was to construct the expressive vector of human tissue factor (TF), and determine its expressive level in stable-transfected human ovarian cancer cell line. The human TFcDNA was obtained from human placenta by RT-PCR and then inserted into eukaryotic expressive vector pcDNA3 to obtain the TF-pcDNA3 recombinant. This recombinant gene was introduced into human ovarian cell line A2780 through transfection mediated by lipofectamine. Stable-transfected cells were screened by G418. The TF expressive levels were detected by RT-PCR and flow cytometry. The results showed that: (1) the constructed product was identified as TF-pcDNA3 recombinant by sequencing. (2) TF was highly expressed not only at transcriptional level in the stable-transfected A2780 cell (transfected cell 3.99 +/- 0.15, 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]. It was concluded that TF gene was successfully cloned, and was introduced into human ovarian cancer cell, and the subline A2780/TF which stably expresses TF at high level was obtained. It will provide good experimental basis for investigating new mechanisms of tumor growth, invasion, metastasis, hypercoagulability, and for exploring a new strategy of gene therapy.


Assuntos
Neoplasias Ovarianas/metabolismo , Proteínas Recombinantes/biossíntese , Tromboplastina/genética , Animais , Linhagem Celular Tumoral , Clonagem Molecular , Feminino , Humanos , Camundongos , Neoplasias Ovarianas/patologia , Tromboplastina/análise , Tromboplastina/biossíntese , Transfecção
15.
Zhonghua Xue Ye Xue Za Zhi ; 24(3): 149-51, 2003 Mar.
Artigo em Zh | MEDLINE | ID: mdl-12697128

RESUMO

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.


Assuntos
Células Endoteliais/efeitos dos fármacos , Fator VII/metabolismo , Oligodesoxirribonucleotídeos/farmacologia , Tromboplastina/biossíntese , Células Cultivadas , Células Endoteliais/metabolismo , Citometria de Fluxo , Humanos , Oligodesoxirribonucleotídeos/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tromboplastina/genética , Transfecção , Fator de Necrose Tumoral alfa/farmacologia , Veias Umbilicais/citologia
16.
Zhonghua Xue Ye Xue Za Zhi ; 25(9): 523-7, 2004 Sep.
Artigo em Zh | MEDLINE | ID: mdl-15569528

RESUMO

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.


Assuntos
Fator VIIa/genética , Neoplasias Ovarianas/patologia , Tromboplastina/genética , Animais , Linhagem Celular Tumoral , Movimento Celular , Clonagem Molecular , Fator VIIa/fisiologia , Feminino , Vetores Genéticos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias Ovarianas/genética , Tromboplastina/fisiologia , Transfecção , Transplante Heterólogo
17.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 12(6): 730-2, 2004 Dec.
Artigo em Zh | MEDLINE | ID: mdl-15631649

RESUMO

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.


Assuntos
Fator VII/análise , Uremia/sangue , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/sangue , Infarto do Miocárdio/etiologia , Diálise Renal , Fatores de Risco , Uremia/complicações , Uremia/terapia
18.
Zhonghua Xue Ye Xue Za Zhi ; 25(3): 143-6, 2004 Mar.
Artigo em Zh | MEDLINE | ID: mdl-15182581

RESUMO

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.


Assuntos
Fator VIIa/metabolismo , Neoplasias Ovarianas/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Tromboplastina/metabolismo , Regulação para Cima , Linhagem Celular Tumoral , Fator VIIa/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Ovarianas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Tromboplastina/genética , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo
19.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 11(1): 66-9, 2003 Feb.
Artigo em Zh | MEDLINE | ID: mdl-12667293

RESUMO

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.


Assuntos
Células Dendríticas/imunologia , Proteínas Associadas aos Microtúbulos/imunologia , Linfócitos T/imunologia , Adulto , Antígenos CD/análise , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Divisão Celular/imunologia , Linhagem Celular , Citotoxicidade Imunológica/imunologia , Células Dendríticas/transplante , Feminino , Citometria de Fluxo , Células HL-60 , Antígenos HLA-DR/análise , Humanos , Imunoterapia Adotiva , Proteínas Inibidoras de Apoptose , Células K562 , Leucemia Monocítica Aguda/tratamento farmacológico , Leucemia Monocítica Aguda/imunologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/imunologia , Leucemia Mielomonocítica Aguda/tratamento farmacológico , Leucemia Mielomonocítica Aguda/imunologia , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/imunologia , Masculino , Proteínas de Neoplasias , Survivina , Células Tumorais Cultivadas , Vacinação/métodos
20.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 10(5): 441-6, 2002 Oct.
Artigo em Zh | MEDLINE | ID: mdl-12513745

RESUMO

The study was designed to investigate annexin II resulting in molecular pathological mechanism of the primary fibrinolysis and establish annexin II vector model for further research on disturbance of coagulation. A target gene was amplified from human umbilical vein endothelial cells (HUVEC) by RT-PCR. Annexin II gene fragment was purified and ligated with molecular biological recombinant technology. The recombinant of plasmid annexin II was transfected into HL-60 cells and its distribution in the cell and structure characteristics of annexin II protein were evaluated by multi-photon excitation laser scanning microscope. By means of flow cytometry (FCM) and Werstern blot technique, the protein expression was qualitatively and quantitatively analyzed. Transfected cells were treated in vitro with annexin II antisense oligonucleotide (AS) targeting to the start site of annexin II cDNA. The results showed that the recombinant pZeoSV2(+)/ANN II was constructed successfully and expressed in HL-60 cells. The protein expression was distributed on the surface of cell by fluorescence assay. After transfection for 48 hours, the cells occurred higher level of expression. The level of the plasmin was significantly enhanced in the present of annexin II. The FCM and Western blot analysis showed the annexin II expression was similar both in transiently and stably transfected in HL-60 cells. Annexin II antisense oligonucletide and McAb significantly inhibited the activity of plasminogen. It was concluded that annexin II plays an important role in the fibrinotysis. Annexin II vector was defined as a expression tool for further studying fibrinolysis and coagulopathy in malignant disease.


Assuntos
Anexina A2/genética , Fibrinólise , Vetores Genéticos , Anexina A2/fisiologia , Endotélio Vascular/química , Endotélio Vascular/citologia , Células HL-60 , Humanos , Oligonucleotídeos Antissenso/farmacologia , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese
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