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1.
Chinese Journal of Cardiology ; (12): 522-526, 2012.
Artigo em Chinês | WPRIM | ID: wpr-326478

RESUMO

<p><b>OBJECTIVE</b>To explore the effects of astragali radix extract on the expressions of matrix metalloproteinase 9 (MMP-9) and the formation of atherosclerotic plaque in aortic atherosclerotic plaques of apolipoprotein E-deficient mice (ApoE-/-).</p><p><b>METHODS</b>Male 8-week-old ApoE-/- mice fed with high fat diet were randomly divided into four groups (n=12 each): control group (saline 0.2 ml/d), atorvastatin group (atorvastatin 10 mg×kg(-1)×d(-1)), low-dose astragali radix extract group (1.25 g×kg(-1)×d(-1)) and high-dose astragali radix extract group (5 g×kg(-1)×d(-1)). After 12 weeks, serum oxLDL was measured by the method of ELISA. The formation of atherosclerotic plaque was determined in HE and oil red O stained aortic slice. The expressions of macrophage and MMP-9 in the aortic plaque were detected by immune fluorescence and immunohistochemistry staining method.</p><p><b>RESULTS</b>Similarly as atorvastatin, astragali radix extract significantly decreased the level of serum oxLDL in ApoE-/-1 mice in a dose-dependent manner. The level of oxLDL in the high-dose astragali radix extract group [(5.2±6.1) µg/ml] was significantly lower than that in the control group [(15.8±5.4) µg/ml, P<0.01]. The area of atherosclerosis plaques was smaller (17.24%±4.22% vs. 49.87%±9.37%, P<0.01) and the penetration degree of plaques in the arterial wall was relieved in the high-dose astragali radix extract group compared to those in the control group (P<0.01). The expressions of Mac3 in atherosclerosis plaques of the high-dose astragali radix extract group was also significantly lower than in the control group (P<0.01). The mean absorbance value of the expression of MMP-9 in the high-dose astragali radix extract group (0.0154±0.0014)was significantly lower than that in the control group (0.0263±0.0065) (P<0.01).</p><p><b>CONCLUSIONS</b>Similar as atorvastatin, astragali radix extract can dose-dependently inhibit the expression of MMP-9 and the formation of the atherosclerotic plaque in ApoE-/- mouse, probably by reducing the serum oxLDL, inhibiting macrophage infiltration, migration and secretion of MMP-9.</p>


Assuntos
Animais , Masculino , Camundongos , Aorta , Apolipoproteínas E , Genética , Astrágalo , Dieta Hiperlipídica , Medicamentos de Ervas Chinesas , Farmacologia , Usos Terapêuticos , Metaloproteinase 9 da Matriz , Metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Placa Aterosclerótica , Tratamento Farmacológico , Metabolismo , Patologia
2.
Chinese Journal of Cardiology ; (12): 531-537, 2011.
Artigo em Chinês | WPRIM | ID: wpr-272205

RESUMO

<p><b>OBJECTIVE</b>Tumor necrosis factor-α (TNF-α) is known to induce changes in endothelial cell morphology and permeability. The aim of this study is to determine the underlying signaling mechanisms involved in these responses.</p><p><b>METHODS</b>Cultured human umbilical vein endothelial cells (HUVECs) were exposed to TNF-α, and HUVEC cytoskeletal changes were evaluated by observing fluorescence of F-actin following ligation with labeled antibodies. Endothelial permeability was detected by measuring the flux of horseradish peroxidase (HRP)-albumin across the EC monolayers. To explore the signaling pathways behind TNF-α-induced changes in HUVEC morphology and permeability, HUVECs were treated with either the Rho GTPase inhibitor Y27632 or the mitogen-activated protein kinases (MAPK) inhibitors PD98059 and SB203580 before TNF-α administration. To further elucidate possible involvement of the RhoA and ERK pathways in TNF-α-induced HUVEC changes, retrovirus-carried recombinant dominant-negative forms and constitutive-activative forms of RhoA, namely T19NRhoA and Q63LRhoA, were pre-infected into HUVECs prior to TNF-α exposure.</p><p><b>RESULTS</b>TNF-α induced F-actin cytoskeleton rearrangement and increased HUVEC permeability in a dose and time-dependent manner. The maximal increase in the HRP-BSA flux (40 ng/ml) was seen in cells exposed to TNF-α at 100 ng/ml after 2 h. Preconditioning of HUVEC monolayer with Y27632 or PD98059 significantly reduced TNF-α induced permeability increase (HRP concentration from 40 ng/ml decreased to 12.5 ng/ml, P < 0.05) and F-actin cytoskeleton rearrangement, HUVEC pre-infection with activated forms of Q63LRhoA increased HUVEC permeability and upregulated pERK compared to GFP infection, while HUVEC pre-infection with inhibited forms of T19NRhoA attenuated the effects of TNF-α on HUVEC permeability.</p><p><b>CONCLUSION</b>These results indicate that TNF-α-induced EC barrier dysfunction and morphological changes of the F-actin via activating RhoA-ERK/MAPK signal pathway.</p>


Assuntos
Humanos , Permeabilidade da Membrana Celular , Células Cultivadas , Citoesqueleto , Metabolismo , Células Endoteliais , Biologia Celular , Metabolismo , Endotélio Vascular , Biologia Celular , Células Endoteliais da Veia Umbilical Humana , Proteínas Quinases Ativadas por Mitógeno , Metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa , Farmacologia , Proteína rhoA de Ligação ao GTP , Metabolismo
3.
Chinese Journal of Applied Physiology ; (6): 297-301, 2010.
Artigo em Chinês | WPRIM | ID: wpr-340167

RESUMO

<p><b>OBJECTIVE</b>To purify the recombinant human cellular repressor of EIA stimulated gene (hCREG)/myc-His glycoprotein and confirm the biological function of hCREG/myc-His which could inhibit the proliferation of human internal thoracic artery smooth muscle cells (HITASY) cultured in vitro.</p><p><b>METHODS</b>The recombinant hCREG/myc-His protein was purified with Ni-NTA column according to 6 x His affinity chromatographic theory. The recombinant hCREG/myc-His protein was desalted by HiTrap Desalting Column. The effect of recombinant hCREG/myc-His glycoprotein of different concentration (0.5 microg/ml, 1 microg/ml and 2 microg/ml) on proliferation of HITASY cells was studied by flow cytometric analysis and the effect of recombinant protein on proliferation of HITASY cells was confirmed by BrdU incorporation method.</p><p><b>RESULTS</b>The recombinant hCREG protein was purified with Ni-NTA column according to 6 x His affinity chromatographic theory. The concentration of recombinant hCREG protein which has been concentrated and desalted was determined to be 1.6 mg/ml and the purity of recombinant protein reached 92%. The protein was identified to be glycosylated. The recombinant hCREG protein was identified to inhibit the proliferation of HITASY cells cultured in vitro and the inhibition effect was stronger in low-dosage group than that in high-dosage group by flow cytometric analysis. The proliferation of HITASY cells cultured in vitro with 2 microg/ml recombinant hCREG protein was inhibited significantly compared with that in control group according to the BrdU incorporation result. There was statistical difference among the groups (P < 0.05).</p><p><b>CONCLUSION</b>The purification of recombinant hCREG/myc-His glycoprotein with biological activity provides an experiment platform for function study and engineering production of hCREG protein.</p>


Assuntos
Humanos , Adenoviridae , Divisão Celular , Células Cultivadas , Glicoproteínas , Glicosilação , Proteínas Recombinantes , Metabolismo , Proteínas Repressoras , Metabolismo
4.
Chinese Journal of Cardiology ; (12): 870-874, 2010.
Artigo em Chinês | WPRIM | ID: wpr-244125

RESUMO

<p><b>OBJECTIVE</b>To observe the dynamic changes of plasma matrix metalloproteinases (MMPs) and investigate the effect of early or delayed percutaneous coronary intervention (PCI) in the presence or absence cilostazol on left ventricle (LV) remodeling in patients with non-ST elevation myocardial infarction (NSTEMI).</p><p><b>METHODS</b>One hundred and sixty-four patients undergoing PCI with NSTEMI were randomized to early PCI (PCI within 24 h) group or delayed PCI group (PCI after 36 h), and patients in both group were further assigned to cilostazol or no cilostazol group. Plasma MMP-2 and MMP-9 concentrations were measured at 2, 4 days and 2 and 4 weeks after PCI. Left ventricular end-diastolic volume (LVEDV), left ventricle ejection fraction (LVEF), left ventricle posterior wall (LVPW) and interventricular septum (IVS) were measured by echocardiography at baseline and 1 year after PCI.</p><p><b>RESULTS</b>MMP-2 concentration at 2 weeks after PCI is higher than that at 2, 4 days and 4 weeks after PCI. MMP-9 concentration at 4 days is higher than that at 2 days, 2 weeks and 4 weeks after PCI. MMP-2 and MMP-9 were significantly lower in cilostazol group compared with that in non-cilostazol group at 4 days, 2 weeks and 4 weeks after NSTEMI (all P < 0.05). Changes of LVEDV and LVEF were significantly less in cilostazol group and early PCI group than that in no cilostazol group and delay PCI group (P < 0.05 or P < 0.01) at 1 year after NSTEMI.</p><p><b>CONCLUSIONS</b>Early PCI and Cilostazol use are associated with less LV remodeling in patients with NSTEMI. Cilostazol attenuated LV remodeling possibly by reducing concentration of MMP-2 and MMP-9 after PCI.</p>


Assuntos
Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Angioplastia Coronária com Balão , Eletrocardiografia , Metaloproteinase 2 da Matriz , Metabolismo , Metaloproteinase 9 da Matriz , Metabolismo , Infarto do Miocárdio , Terapêutica , Tetrazóis , Usos Terapêuticos , Fatores de Tempo , Resultado do Tratamento , Remodelação Ventricular
5.
Chinese Journal of Applied Physiology ; (6): 244-249, 2009.
Artigo em Chinês | WPRIM | ID: wpr-356285

RESUMO

<p><b>AIM</b>To construct short hairpin RNA (shRNA) eukaryotic expression vectors targeting TGF-beta1 for further research on the effects of TGF-beta1 on vasculogenesis and angiogenesis.</p><p><b>METHODS</b>Three pairs of siRNA target sequences coding from the mRNA of TGF-beta1 gene were designed and three pairs of nucleotides were synthesized. After annealing, the double-strand DNA products were ligated into the pEN_mH1c entry vector, and in turn into the shRNA eukaryotic expression vector pDS_hpEy labled by GFP through the LR recombination reaction. After sequencing successfully, the three resulting TGF-beta1 shRNA expression vectors were transfected into the mouse fibroblast cell line (NIH/3T3), and then cell clones stably expressing TGF-beta1 shRNA were screened. Reverse Transcript-Polymerase Chain Reaction (RT-PCR) and Western blot were used to detect the mRNA and protein expression.</p><p><b>RESULTS</b>RT-PCR and Western blot showed that one of the TGF-beta1 shRNA expression vectors pDS_Tc downregulated TGF-pl mRNA and protein expression markedly in NIH/3T3 cells.</p><p><b>CONCLUSION</b>ShRNA eukaryotic expression vectors targeting TGF-beta1 are successfully constructed which can be used for further investigation on the mechanism through which TGF-beta1 regulates vasculogenesis and angiogenesis.</p>


Assuntos
Animais , Camundongos , Vetores Genéticos , Proteínas de Fluorescência Verde , Genética , Metabolismo , Células NIH 3T3 , Neovascularização Fisiológica , Interferência de RNA , RNA Mensageiro , Genética , Metabolismo , RNA Interferente Pequeno , Genética , Transfecção , Fator de Crescimento Transformador beta1 , Genética , Metabolismo
6.
Journal of Southern Medical University ; (12): 1073-1075, 2008.
Artigo em Chinês | WPRIM | ID: wpr-270206

RESUMO

<p><b>OBJECTIVE</b>To investigate the changes of high-sensitive C-reactive protein (hs-CRP) and monocyte chemotactic factor-1 (MCP-1) following percutaneous coronary interventional procedures (PCI) in patients with coronary artery disease (CAD), and evaluate the impact of PCI on the inflammatory indices and postoperative vascular restenosis.</p><p><b>METHODS</b>This study involved 80 patients undergoing PCI procedures for CAD compromising a single coronary artery. Forty healthy individuals with normal findings by coronary angiography were selected as the control group. Before and after PCI or coronary angiography, plasma hs-CRP and MCP-1 were measured in all the subjects by immunonephelometry and enzyme-linked immunosorbant assay (ELISA), respectively.</p><p><b>RESULTS</b>In the CAD patients, the plasma hs-CRP level was significantly elevated after PCI as compared with the preoperative level (2.37-/+0.56 microg/L vs 1.59-/+0.41 microg/L, P<0.01), whereas in the control group, the hs-CRP level underwent no significant changes after coronary angiography (1.18-/+0.37 microg/L vs the preoperative level of 1.13-/+0.32 microg/L, P>0.05). PCI procedures also resulted in significant elevation of plasma MCP-1 level in the CAD patients (26.04-/+5.43 pg/L vs the preoperative level of 18.07-/+4.30 pg/L, P<0.01), but in the control group, MCP-1 showed no significant variation after coronary angiography (9.80-/+2.64 pg/L vs the preoperative level of 9.63-/+2.52 pg/L, P>0.05).</p><p><b>CONCLUSION</b>Plasma hs-CRP and MCP-1 are elevated in CAD patients following PCI procedures, but their roles in the vascular restenosis following the procedures need further investigation.</p>


Assuntos
Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Angioplastia Coronária com Balão , Proteína C-Reativa , Quimiocina CCL2 , Sangue , Doença da Artéria Coronariana , Sangue , Terapêutica
7.
Chinese Journal of Cardiology ; (12): 195-198, 2008.
Artigo em Chinês | WPRIM | ID: wpr-243817

RESUMO

<p><b>OBJECTIVE</b>To investigate the relationship between matrix metalloproteinase (MMP) 1 gene -519A/G polymorphism and the risk of coronary heart disease (CHD) in Northern Chinese Han population.</p><p><b>METHODS</b>A total of 517 patients with CHD and 380 healthy adults diagnosed by coronary angiography were genotyped by polymerase chain reaction-restriction fragment length polymorphism and DNA sequence technology for the -519A/G polymorphism in MMP1 gene.</p><p><b>RESULTS</b>(1) The frequency of AA genotype was significantly higher in patients with CHD than that in controls [67.70% (350/517) vs. 40.26% (153/380), OR = 1.64, P < 0.001, 95%CI: 1.44 - 1.86]. People carrying A allele had increased risk for CHD (OR = 1.49, P < 0.001, 95%CI: 1.33 - 1.69). (2) The frequency of AA genotype was higher in patients with acute coronary syndrome (ACS) than patients with stable angina pectoris [68.81% (278/404) vs. 51.76% (44/85), P < 0.01, 95%CI: 1.04 - 1.27]. The A allele carriers were more likely to develop ACS (OR = 1.11, 95%CI: 1.01 - 1.21, P < 0.05).</p><p><b>CONCLUSION</b>Our data shows MMP1 gene -519A/G polymorphism is associated with the risk of CHD, and A allele carriers are more susceptible for CHD in Northern Chinese Han population.</p>


Assuntos
Adulto , Idoso , Idoso de 80 Anos ou mais , Humanos , Masculino , Pessoa de Meia-Idade , Povo Asiático , Genética , Doença das Coronárias , Etnologia , Genética , Predisposição Genética para Doença , Metaloproteinase 1 da Matriz , Genética , Polimorfismo de Nucleotídeo Único
8.
Chinese Journal of Applied Physiology ; (6): 385-390, 2008.
Artigo em Chinês | WPRIM | ID: wpr-252761

RESUMO

<p><b>AIM</b>To observe expression regularity of SMalpha-actin, SM22alpha, myocardin and SMMHC during early embryonic vascular development, and to initially investigate the differentiation effect of platelet derived growth factor-BB (PDGF-BB) on vascular smooth muscle cells (VSMCs) during that period.</p><p><b>METHODS</b>Murine embryonic stem cell line expressing the enhanced green fluorescent protein (GFP) under the transcriptional control of the smooth-muscle-specific SM22alpha promoter was used to make embryoid bodies,and to analyze the expression regularity of SMalpha-actin, SM22alpha, myocardin and SMMHC by immunofluorescence stainings, RT-PCR and Western blot. Then AG1296 (PDGF receptor inhibitor) 0 micro-mol/L(control group), 10 micromol/L and 50 micromol/L were used to treat EBs respectively in order to analyze the differences of SMa-actin, SM22alpha, myocardin and SMMHC at gene and protein levels among the three groups.</p><p><b>RESULTS</b>SMalpha-actin, myocardin, SM22alpha and SMMHC expression in EBs were found to begin at day 0 (ESCs), 8, 11, 13 respectively during early embryonic vascular development. There were no clear differences in SMa-actin, SM22alpha, myocardin and SMMHC protein expression and SM22alpha, myocardin and SMMHC mRNA level among the three groups of different concentrations of AG1296.</p><p><b>CONCLUSION</b>A spontaneous VSMCs differentiation occurs during EBs development, SMalpha-actin is the first to be detected,the following are myocardin, SM22a and SMMHC. PDGF-BB may not be indispensable for the regulation of expression of VSMCs markers during early EBs differentiation.</p>


Assuntos
Animais , Camundongos , Actinas , Genética , Metabolismo , Biomarcadores , Metabolismo , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias , Biologia Celular , Metabolismo , Proteínas dos Microfilamentos , Genética , Metabolismo , Proteínas Musculares , Genética , Metabolismo , Músculo Liso Vascular , Biologia Celular , Proteínas Nucleares , Genética , Metabolismo , Proteínas Proto-Oncogênicas c-sis , Metabolismo , RNA Mensageiro , Genética , Metabolismo , Transativadores , Genética , Metabolismo
9.
Chinese Journal of Cardiology ; (12): 788-792, 2007.
Artigo em Chinês | WPRIM | ID: wpr-307198

RESUMO

<p><b>OBJECTIVE</b>To evaluate the interaction of atorvastatin or pravastatin with clopidogrel on platelet activation and aggregation function in patients with acute coronary syndromes (ACS) undergoing coronary stenting.</p><p><b>METHODS</b>Between April and December 2006, a total of 150 hospitalized ACS patients undergoing coronary stenting were randomized to receive atorvastatin (n = 50), pravastatin (n = 50) or no statin (n = 50) one day post procedure. All patients received standard antiplatelet treatment including aspirin 300 mg/d and loading dose 300 mg of clopidogrel followed by maintenance dose 75 mg/d. The expressions of CD62P and PAC-1 and the maximal platelet aggregation rate (MPAR) induced by 20 micromol/L ADP were measured at day 1 before statin therapy (baseline) and day 3 after procedure.</p><p><b>RESULTS</b>Baseline clinical characteristics and levels of CD62P, PAC-1 and MPAR at the baseline were comparable among three groups. After 3-day statin treatment, the changes of CD62P [(4.69 +/- 16.78)% vs. (1.35 +/- 10.86)% vs. (2.97 +/- 10.21)%], PAC-1 [(12.78 +/- 22.07)% vs. (8.01 +/- 21.23)% vs. (10.65 +/- 21.39)%] and MPAR [(5.44 +/- 18.68)% vs. (7.15 +/- 19.59)% vs. (3.76 +/- 23.42)%] among three groups were not significantly different (all P > 0.05). Subgroup analysis showed that DeltaCD62P [(7.50 +/- 19.35)% vs. (3.24 +/- 11.18)% vs. (2.53 +/- 8.87)%], DeltaPAC-1 [(13.40 +/- 24.62)% vs. (11.28 +/- 19.90)% vs. (10.11 +/- 21.29)%] and DeltaMPAR [(7.56 +/- 19.11)% vs. (7.87 +/- 23.60)% vs. (6.75 +/- 23.30)%] in ACS patients were also similar among three groups (all P > 0.05).</p><p><b>CONCLUSION</b>Neither atorvastatin nor pravastatin attenuates the antiplatelet function of clopidogrel in ACS patients early post coronary stenting.</p>


Assuntos
Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Síndrome Coronariana Aguda , Tratamento Farmacológico , Terapêutica , Angioplastia Coronária com Balão , Atorvastatina , Interações Medicamentosas , Ácidos Heptanoicos , Usos Terapêuticos , Inibidores de Hidroximetilglutaril-CoA Redutases , Usos Terapêuticos , Agregação Plaquetária , Inibidores da Agregação Plaquetária , Usos Terapêuticos , Pravastatina , Usos Terapêuticos , Estudos Prospectivos , Pirróis , Usos Terapêuticos , Ticlopidina , Usos Terapêuticos
10.
Acta Physiologica Sinica ; (6): 207-216, 2006.
Artigo em Chinês | WPRIM | ID: wpr-265463

RESUMO

To investigate the role and mechanism of Rac1 protein in the process of the human umbilical vein endothelial cell (HUVEC) senescence, we used hypoxia as a model for modulating HUVECs entering replicative senescence in vitro. Premature senescence of HUVECs was evidenced by detecting the SA-beta-Gal activity and PAI-1 expression. Meanwhile, cell cycle distribution and cell proliferation rate were investigated by flow cytometry assay and BrdU staining. The results indicated that the HUVECs became enlarged and flattened, both SA-beta-Gal activity and PAI-1 expression increased obviously, while cell proliferation was inhibited and G(1) phase cell cycle arresting occurred when HUVECs were treated with continued hypoxia for 96 h. Accompanied with these changes, the expression of activated Rac1 increased obviously in cells after hypoxia. All these observations suggested that endothelial senescence could be induced by continued hypoxia and it might correlate with the activity of Rac1. To further define the relationship between Rac1 and HUVEC senescence, HUVECs were transiently infected with the constitutively active form of Rac1 (V12Rac1) or dominant negative form of Rac1 (N17Rac1) using retrovirus vector pLNCX-V12Rac1 or pLNCX-N17Rac1. We observed the changes of these three kinds of HUVECs (HUVECs, N17Rac1-HUVECs, V12Rac1-HUVECs) after hypoxia for 48 h and 96 h, the expression and localization of serum response factor (SRF), which is one of the downstream signal molecules of Rac1, were also investigated. The results obtained indicated that after continued hypoxia for 48 h, HUVECs infected by V12Rac1 showed obvious senescence accompanied with SA-beta-Gal activation, PAI-1 expression increase, G(1) phase arrest and cell proliferation inhibition which were similar to HUVECs after continued 96-hour hypoxia treatment, while the senescence of HUVECs infected by N17Rac1 was significantly inhibited even if the cells were exposed to hypoxia for more than 96 h. All the results identified that the activation of Rac1 might accelerate HUVEC senescence induced by hypoxia and that inactivation of Rac1 could partly block the cell senescence. To further investigate the mechanism of HUVEC senescence induced by Rac1, we detected the expression of total SRF (tSRF) and nuclear SRF (nSRF) in these three kinds of HUVECs by immunofluorescent analysis and Western blot assay after hypoxia. The results showed that the expression of nSRF decreased obviously and the nuclear translocation of SRF was inhibited in HUVECs infected by V12Rac1 compared with those in the normal HUVECs. In contrast, the expression of nSRF increased obviously in the HUVECs infected by N17Rac1. These results suggest that activation of Rac1 accelerates endothelial cell senescence and inhibition of Rac1 activity prevents HUVECs from entering senescence induced by hypoxia, while the nuclear translocation of SRF regulated by Rac1 might play an important role in the process of senescence.


Assuntos
Humanos , Hipóxia Celular , Células Cultivadas , Senescência Celular , Fisiologia , Células Endoteliais da Veia Umbilical Humana , Biologia Celular , Inibidor 1 de Ativador de Plasminogênio , Genética , Metabolismo , Fator de Resposta Sérica , Genética , Metabolismo , beta-Galactosidase , Metabolismo , Proteínas rac1 de Ligação ao GTP , Metabolismo
11.
Acta Physiologica Sinica ; (6): 324-330, 2006.
Artigo em Chinês | WPRIM | ID: wpr-265448

RESUMO

To investigate the effects and molecular mechanisms of the cellular repressor of E1A-stimulated genes (CREG) on the apoptosis of vascular smooth muscle cells (VSMCs), the human internal thoracic artery-Shenyang (HITASY) cells were infected with sense-CREG [pLNCX(2)(+)/CREG] and antisense-CREG [pLXSN(-)/CREG] retrovirus respectively. The stably infected cells were obtained by screening the G418-resistant clones. DAPI nuclei staining and Annexin V/PI FASC assay indicated that over-expression of CREG in HITASY cells infected with pLNCX(2) (+)/CREG inhibited VSMC apoptosis induced by serum deprivation, accompanied with decreased expression of caspase-9 mRNA detected by RT-PCR. Furthermore, Western blot analysis showed that p38 mitogen activated protein kinase (p38 MAPK) expression and activation were significantly enhanced in HITASY cells infected with pLNCX(2) (+)/CREG. The inhibition of CREG protein expression in cells infected with pLXSN(-)/CREG promoted the VSMC spontaneous apoptosis, as well as down-regulated p38 MAPK expression and activation, when cells were cultured with 10% fetal bovine serum (FBS) mediums. These results implicate that the CREG protein has the ability to regulate VSMC apoptosis in which the activation of p38 MAPK is possibly involved. To further identify the role of p38 MAPK in VSMC apoptosis, SB203580, a specific inhibitor of p38 MAPK, was used to inhibit p38 MAPK activity. When p38 MAPK signaling pathway was blocked, the effects that over-expression of CREG protein inhibited VSMC apoptosis disappeared. Taken together, the present work indicates that over-expression of CREG protein inhibits VSMC apoptosis, and this inhibitory effect is partly mediated by p38 MAPK signaling pathway.


Assuntos
Humanos , Apoptose , Fisiologia , Caspase 9 , Metabolismo , Células Cultivadas , Músculo Liso Vascular , Biologia Celular , Miócitos de Músculo Liso , Metabolismo , Proteínas Repressoras , Genética , Fisiologia , Transdução de Sinais , Fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno , Metabolismo
12.
Acta Physiologica Sinica ; (6): 295-302, 2005.
Artigo em Chinês | WPRIM | ID: wpr-334172

RESUMO

RhoA is one of the main members of RhoGTPase family involved in cell morphology, smooth muscle contraction, cytoskeletal microfilaments and stress fiber formation. It has been demonstrated that RhoA modulates endothelial cell permeability by its effect on F-actin rearrangement, but the molecular mechanism of rearrangement of actin cytoskeleton remains unclear. Recent studies prove that RhoA/Rho kinase regulates smooth muscle specific actin dynamics by activating serum response factor (SRF)-dependent transcription. To further investigate the molecular mechanism of the rearrangement of vascular endothelial cell actin cytoskeleton, we explored the relationship between the activation of SRF and F-actin rearrangement induced by RhoA in human umbilical vein endothelial cells (HUVECs). HUVECs were infected with the constitutively active forms of RhoA (Q63LRhoA) or the dominant negative forms of RhoA(T19NRhoA) using retrovirus vector pLNCX-Q63LRhoA or pLNCX-T19NRhoA, the positive clone was obtained by G418 selection. The expression and distribution of SRF in normal and infected cells were evaluated by immunohistochemistry and Western blot in complete medium and in serum-free medium. The effect of F-actin polymerization was detected by Rhodamine-Phalloidine staining. Infection of PLNCX-Q63LRhoA induced F-actin rearrangement and stress fiber formation in HUVECs, as well as enhanced the expression of SRF in the nuclei. In contrast, the cells infected with T19NRhoA showed no distinct changes. With serum deprivation, the expression of SRF increased obviously in both normal and infected HUVECs, but the subcellular localization of SRF was evidently different. In HUVECs, the localization of SRF was in the nuclei after 3 d with serum deprivation, but it was redistributed outside the nuclei after 5 d with serum deprivation. In cells infected with Q63LRhoA, the immunolocalization of SRF was always in the nuclei compared with HUVECs infected with T19NRhoA, which was almost always localized in the cytoplasm. In HUVECs, the rearrangement of F-actin and formation of stress fiber increased after 3 d with serum deprivation, but appeared decreased and unpolymerized after 5 d with serum deprivation. The polymerization of F-actin and the formation of stress fiber in HUVECs infected with Q63LRhoA kept during the period of serum-free culture, whereas the rearrangement of F-actin in cells infected with T19NRhoA was not found. These results suggest that RhoA influences endothelial F-actin rearrangement in part by regulating the expression and subcellular localization of SRF.


Assuntos
Humanos , Actinas , Genética , Citoesqueleto , Metabolismo , Endotélio Vascular , Biologia Celular , Metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Serina-Treonina Quinases , Metabolismo , Fator de Resposta Sérica , Genética , Veias Umbilicais , Biologia Celular , Quinases Associadas a rho , Proteína rhoA de Ligação ao GTP , Fisiologia
13.
Chinese Journal of Medical Genetics ; (6): 661-664, 2005.
Artigo em Chinês | WPRIM | ID: wpr-279975

RESUMO

<p><b>OBJECTIVE</b>To identify the relationship between p53-dependent apoptosis associated genes and tumor metastasis.</p><p><b>METHODS</b>mRNA differential display (mRNA DD) was adopted for gene cloning after the different metastatic potential lung cancer cell lines were infected by Adv-p53 (a reconstructed adenovirus encoding wild type p53 gene). RT-PCR, Northern blot and Western blot assays were used to confirm the result from mRNA DD.</p><p><b>RESULTS</b>After induction by p53 gene, the ANNEXIN A2 gene had differential expression in the cell lines; its level was down regulated in all the cells infected by Adv-p53 gene, especially in the Anip973 cell lines with high metastatic potential. RT-PCR, Northern blot and Western blot assays confirmed the consequence.</p><p><b>CONCLUSION</b>The experimental data suggest that the ANNEXIN A2 gene may relate to cellular apoptosis induced by p53 gene. The affirmative relationship between ANNEXIN A2 gene and p53 needs further investigation.</p>


Assuntos
Humanos , Adenoviridae , Genética , Anexina A2 , Genética , Metabolismo , Apoptose , Genética , Sequência de Bases , Northern Blotting , Western Blotting , Linhagem Celular Tumoral , Clonagem Molecular , Regulação Neoplásica da Expressão Gênica , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Proteína Supressora de Tumor p53 , Genética , Metabolismo
14.
Acta Academiae Medicinae Sinicae ; (6): 149-152, 2003.
Artigo em Chinês | WPRIM | ID: wpr-278109

RESUMO

<p><b>OBJECTIVE</b>To study the growth inhibitory effects of p21WAF1 and p53 overexpression in human lung adenocarcinoma cell line.</p><p><b>METHODS</b>The p21WAF1 and p53 gene were transfected respectively into a human lung adenocarcinoma cell line, GLC-82. Flow cytometry (FLC), transmission electron microscopy (EM) and TUNEL technique were used to evaluate cell growth and identify apoptosis.</p><p><b>RESULTS</b>The GLC-82 transfected by p21 plasmid showed increased cell number in G1 phase of cell cycle, decreased proliferation potential and decreased cloning efficiency. Apoptosis have not been detected neither on EM nor by TUNEL technique, whereas the GLC-82 infected by Ad-p53 showed significantly decreased proliferation potential and some of them even died, in addition apoptosis was confirmed by TUNEL technique.</p><p><b>CONCLUSION</b>The results indicate that p21WAF1 and p53 can inhibit proliferation; p53 also can induce apoptosis of lung adenocarcinoma cell. Therefore, these two genes should have a wide application in gene therapy of tumors in future.</p>


Assuntos
Humanos , Adenocarcinoma , Genética , Metabolismo , Patologia , Apoptose , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas , Genética , Expressão Gênica , Neoplasias Pulmonares , Genética , Metabolismo , Patologia , Proteína Supressora de Tumor p53 , Genética
15.
Chinese Journal of Medical Genetics ; (6): 409-412, 2003.
Artigo em Chinês | WPRIM | ID: wpr-329448

RESUMO

<p><b>OBJECTIVE</b>To evaluate the potential of p53 gene therapy for lung cancer in nude mice.</p><p><b>METHODS</b>Two lung adenocarcinoma cell lines L-18 and 95D were infected with adenovirus encoding wild-type p53 gene pAdCMV -p53 (Ad-p53 ) in vitro and in vivo. The antitumor effect of wild type p53 gene was assessed by cell growth curve, reverse transcriptase polymerase chain reaction (RT-PCR) analysis and TUNEL staining methods.</p><p><b>RESULTS</b>The p53-specific growth inhibition and apoptosis of tumor cells were observed in both cell lines in vitro. By RT-PCR analysis, the increasing expression of p21 gene but not of p16 gene after p53 gene infection suggested that p21 gene played an important role in p53 gene induced cell apoptosis. The in vivo study revealed that celiac injection of p53 gene significantly inhibited the tumorigenesis in 95D and L-18 cells in nude mice. However, no obvious inhibition of tumorigenesis was observed after subcutaneous injection of p53 gene in L-18 cell line, compared with the inhibition noted in 95D cell line.</p><p><b>CONCLUSION</b>The results showed the adenovirus-mediated antitumor therapy by means of p53 gene infection might be a potential way to inhibit cancer growth and induce tumor cell apoptosis.</p>


Assuntos
Animais , Camundongos , Adenocarcinoma , Genética , Patologia , Adenoviridae , Genética , Apoptose , Genética , Divisão Celular , Genética , Inibidor p16 de Quinase Dependente de Ciclina , Genética , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos , Genética , Marcação In Situ das Extremidades Cortadas , Neoplasias Pulmonares , Genética , Patologia , Camundongos Nus , Transplante de Neoplasias , Proteína Oncogênica p21(ras) , Genética , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53 , Genética
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