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1.
Chinese Medical Journal ; (24): 205-211, 2020.
Article Dans Anglais | WPRIM | ID: wpr-781619

Résumé

BACKGROUND@#Angiogenesis and hypoxia-inducible factor 1α (HIF-1α) play major roles in solid tumors. This study aimed to establish a longitudinal and multimodal imaging model for in vivo evaluation of HIF1α and angiogenesis in breast cancer.@*METHODS@#By transfection of a 5 hypoxia-responsive element (HRE)/green fluorescent protein (GFP) plasmid, the cell line Ca761-hre-gfp was established, which emitted green fluorescence triggered by HIF-1α under hypoxia. The cells were subjected to CoCl2-simulated hypoxia to confirm the imaging strategy. We grew Ca761-hre-gfp cells in the left rear flanks of twelve 615 mice. Experiments were conducted on days 4, 9, 15, and 19. For in vivo analysis, Ca761-hre-gfp subcutaneous allografted tumors were imaged in vivo using contrast-enhanced ultrasound (CEUS) and fluorescence imaging (FLI) during tumor development. The tumor size, CEUS peak intensity, and FLI photons were measured to evaluate tumor growth, angiogenesis, and HIF-1α activity, respectively. After each experiment, three mice were randomly sacrificed and tumor specimens were collected to examine HIF-1α activity and the microvessel density (MVD).@*RESULTS@#In vitro, both green fluorescence and HIF-1α expression were detected in Ca761-hre-gfp cells treated with CoCl2, indicating the suitability of the cells to detect HIF-1α activity. In vivo, HIF-1α activity first increased and then decreased, which was significantly correlated with angiogenic changes (r = 0.803, P = 0.005). These changes were confirmed by immunohistochemical staining of HIF-1α and MVD.@*CONCLUSIONS@#The findings validated the Ca761-hre-gfp murine allograft model for reliable evaluation of HIF-1α activity and angiogenesis longitudinally using both molecular and pre-clinical non-invasive imaging modalities. The cell line may be useful for studies of anti-HIF pathway therapies.

2.
Acta Academiae Medicinae Sinicae ; (6): 656-661, 2015.
Article Dans Chinois | WPRIM | ID: wpr-289929

Résumé

<p><b>OBJECTIVE</b>To investigate the correlation of contrast-enhanced pattern with expression of hypoxia inducible factor-1α (HIF-1α) and microvessel density (MVD) in mice breast cancer.</p><p><b>METHODS</b>A total of 22 mice were implanted with breast cancer cells (Ca761) subcutanously in the thigh. The tumors were examined with conventional ultrasound and contrast-enhanced ultrasound (CEUS) on days 4,6,7,8,9,10,and 11 after implantation and then sacrificed. Three or four mice were included each time. Expressions of HIF-1α and MVD in cancer tissues were detected immunohistochemically. Correlation of contrast-enhanced patterns with expression of HIF-1α and MVD in breast cancer was analyzed.</p><p><b>RESULTS</b>Mice were divided into 3 groups according to the tumor volume:group 1 (volume<0.05 cm(3),n=5),group 2 (volume 0.05-0.75 cm(3),n=9),and group 3 (volume>0.75 cm(3),n=8). The CEUS pattern was different in different groups:four mice in group 1 presented as type 1 (peripheral ring enhancement with no enhancement within the tumor) and 1 case presented as type 2 (peripheral ring enhancement with deep penetration). Most mice in group 2 presented as type 3 (homogeneous or heterogeneous enhancement in the whole tumor,n=5). In group 3,most mice presented as type 4 (peripheral ring enhancement with focal nodular enhancement within the tumor,n=7). Contrast-enhanced pattern was significantly different in different volume groups (P<0.01). Enhanced pattern (type 1-4) was closely correlated with tumor volume (r=0.841,P<0.05). The expression of HIF-1α was negatively correlated with enhanced patterns (type 1-4) (r=-0.596,P=0.003),but not with tumor volume (P>0.05). There was no significant difference in MVD values between different enhanced patterns (type 1-4),and there was no correlation between the MVD and tumor volumes (P>0.05).</p><p><b>CONCLUSION</b>CEUS can be used as a noninvasive tool to monitor tumor angiogenesis in tumor and the enhanced patterns may reflect the expression of HIF-1α inside the tumor.</p>


Sujets)
Animaux , Souris , Tumeurs du sein , Lignée cellulaire tumorale , Produits de contraste , Sous-unité alpha du facteur-1 induit par l'hypoxie
3.
Chinese Journal of Pathology ; (12): 330-335, 2013.
Article Dans Chinois | WPRIM | ID: wpr-233459

Résumé

<p><b>OBJECTIVE</b>To investigate the effect of the selective PI3K inhibitor and MEK inhibitor on KRAS and PTEN co-mutated non-small cell lung cancer cell line NCI-H157 and the relevant mechanisms.</p><p><b>METHODS</b>NCI-H157 was cultured routinely and treated with different concentrations of the two inhibitors. Cell proliferation was detected by MTT cell cycle assay. Based on the MTT results the cells were divided into four groups: the control group, PI3K inhibitor group (GDC-0941, 0.5 and 5.0 µmol/L), combination group I (0.5 µmol/L AZD6244 + 0.5 µmol/L GDC-0941) and combination group II (5.0 µmol/L AZD6244 + 5.0 µmol/L GDC-0941). Colony formation assay was performed to detect colony formation efficiency. The cell cycle and apoptosis were analyzed by flow cytometry. The expression of protein related to apoptosis was tested with Western blot.</p><p><b>RESULTS</b>Cell growth was inhibited by the two inhibitors. Combination groups led to stronger cell proliferation inhibition: combination group Ishowed synergistic effect of their actions and combination group II showed an additive effect; in both groups, there were decreased colony number [(77.2 ± 1.54)/well vs (61.50 ± 2.12)/well, P < 0.01] and [(51.00 ± 4.00)/ well vs (22.50 ± 3.53)/well, P < 0.01]; and enhanced apoptotic ratios [(18.30 ± 0.82)% vs (21.32 ± 0.56)%, P < 0.01] and [(27.14 ± 1.58)% vs (42.45 ± 4.42)%, P < 0.01]. In addition, compared to the PI3K inhibitor alone group, the NCI-H157 cells in the combination groups showed increased G0/G1 phase and decreased S phase (P < 0.01). Western blotting showed that the combination groups demonstrated significantly decreased expression of cyclin D1 and cyclin B1, increased p21 and cleaved PARP and decreased bcl-2/bax ratio, compared to the PI3K inhibitor only group.</p><p><b>CONCLUSION</b>The combined inhibition of PI3K (AZD6244) and MEK (GDC-0941) has synergistic effects on the proliferation of NCI-H157 cells, but such effects appear to be in a dose-dependent manner.</p>


Sujets)
Humains , Apoptose , Benzimidazoles , Pharmacologie , Carcinome pulmonaire non à petites cellules , Génétique , Anatomopathologie , Cycle cellulaire , Lignée cellulaire tumorale , Prolifération cellulaire , Cycline B1 , Métabolisme , Cycline D1 , Métabolisme , Relation dose-effet des médicaments , Synergie des médicaments , Indazoles , Pharmacologie , Tumeurs du poumon , Génétique , Anatomopathologie , Mitogen-Activated Protein Kinase Kinases , Métabolisme , Mutation , Phosphohydrolase PTEN , Génétique , Phosphatidylinositol 3-kinases , Métabolisme , Poly(ADP-ribose) polymerases , Métabolisme , Protéines proto-oncogènes , Génétique , Protéines proto-oncogènes c-bcl-2 , Métabolisme , Protéines proto-oncogènes p21(ras) , Métabolisme , Transduction du signal , Sulfonamides , Pharmacologie , Protéine Bax , Métabolisme , Protéines G ras , Génétique
4.
Chinese Journal of Oncology ; (12): 486-490, 2013.
Article Dans Chinois | WPRIM | ID: wpr-267515

Résumé

<p><b>OBJECTIVE</b>Mouse tumors were subcutaneously transplanted into different mouse strains and their growth and metastatic properties were checked, to explore the possibility of establishing animal tumor models in different mouse strains other than their normal host strains.</p><p><b>METHODS</b>Seven mouse tumor cell lines: H22, S180, U14, FC, Ca761, SMG-A and DCS were transplanted into C57BL/6J, ICR or KM mice, and their tumorigenicity, growth and metastasis were recorded and analyzed.</p><p><b>RESULTS</b>The tumor formation rate of H22 cells in both the C57BL/6J and ICR mice was 100%, but the growth of H22 tumors was significantly faster in the C57BL/6J (2.8 ± 0.4)g than in the ICR mice (1.5 ± 0.5)g at the 17th day after transplantation (P<0.001). The S180 tumors grew stably in C57BL/6J mice and the tumor formation rate was 100%. The U14 inoculated into C57BL/6J and KM mice showed both lymphatic and lung metastasis and formed significantly larger tumors in KM mice [(12.6 ± 3.4)g] than that in the C57BL/6J mice [(10.2 ± 2.2)g] on the 32rd day after transplantation (P = 0.002). Transplantation of FC, Ca761, and SMG-A did not form tumors or the tumors were completely regressed later in C57BL/6J mice. DCS cells formed tumors in C57BL/6J mice, but some of the tumors regressed. The retained tumors were passaged in C57BL/6J mice, and the substrain DCS-C57 cells was established which showed stable growth and had a 100% tumor formation rate and 100% lung metastasis rate in C57BL/6J mice.</p><p><b>CONCLUSIONS</b>Cross-strain transplanted tumors can be successfully established by inoculation of poorly differentiated and highly malignant tumor cells into different mouse strains. Some highly immunogenic tumor cells may form tumor, however, the tumors are regressed later, and can not establish cross-strain transplanted tumors in other mouse strains. Stable transplanted tumor models can be obtained from the partially regressed tumors after continuous passages in vivo.</p>


Sujets)
Animaux , Femelle , Mâle , Souris , Lignée cellulaire tumorale , Tumeurs du poumon , Anatomopathologie , Métastase lymphatique , Souris de lignée C57BL , Souris de lignée ICR , Régression tumorale spontanée , Anatomopathologie , Transplantation tumorale , Tumeurs expérimentales , Classification , Anatomopathologie , Transplantation hétérologue , Charge tumorale
5.
Chinese Journal of Pathology ; (12): 842-847, 2010.
Article Dans Chinois | WPRIM | ID: wpr-295130

Résumé

<p><b>OBJECTIVE</b>To investigate the role that E-cadherin (E-cad) plays on cell adhesion and proliferation of human breast carcinoma.</p><p><b>METHODS</b>E-cad expression vector was transfected into an E-cad-negative human breast carcinoma MDA-MB-231 cells. G418 was used to screen positive clones. E-cad, β-catenin (β-cat) and cyclin D1 expressions of these clones were confirmed by Western blot. Their cell-cell and cell-matrix adhesion abilities were detected. E-cad/β-catenin interaction was confirmed by immunoprecipitation. Cell proliferation was evaluated by MTT. Cell apoptosis was analyzed by flow cytometry. Direct two-step immunocytochemistry was used to detect the localization of β-cat.</p><p><b>RESULT</b>E-cad(+) cell strains Ecad-231-7 and Ecad-231-9 were established. When cultured in ultra-low-binding dishes Ecad-231 cells grow in suspension while Ecad-231-7 and Ecad-231-9 cells grow in large clamps. When co-cultured with HCT116 cells, the average adhesion rates at 30 min are 39.0%, 60.0% and 59.5% for MDA-MB-231, Ecad-231-7 and Ecad-231-9 respectively. The average detachment rates by EDTA for 5 min are 37.4%, 4.2% and 7.4% respectively. So E-cad expression enhanced hemotypic and heterotypic cell-cell adhesion and cell-matrix adhesion. Forced exogenously expressed E-cad could combine with endogenous β-cat, whereas down stream cyclin D1 expression was significantly decreased, as evidenced by Western blot. The rates of cell apoptosis of MDA-MB-231, Ecad-231-7 and Ecad-231-9 were 1.8%, 2.0% and 2.1%. Expression of E-cad had no obvious effect on the apoptosis of tumor cells with regular culture. β-cat increased in the cytoplasma.</p><p><b>CONCLUSIONS</b>Two monoclonal tumor cell strains (Ecad-231-7 and Ecad-231-9) stably expressing E-cad were successfully established. E-cad could enhance adhesion and inhibit proliferation of human breast carcinoma cells through a pathway involving β-cat and cyclin D1.</p>


Sujets)
Femelle , Humains , Apoptose , Tumeurs du sein , Métabolisme , Anatomopathologie , Cadhérines , Génétique , Métabolisme , Physiologie , Adhérence cellulaire , Lignée cellulaire tumorale , Prolifération cellulaire , Cycline D1 , Métabolisme , Vecteurs génétiques , Plasmides , Transfection , bêta-Caténine , Métabolisme
6.
Chinese Journal of Pathology ; (12): 110-116, 2009.
Article Dans Chinois | WPRIM | ID: wpr-319776

Résumé

<p><b>OBJECTIVE</b>To establish a green-fluorescent protein (GFP) labeled tumor metastasis model and to evaluate its biological characteristics.</p><p><b>METHODS</b>Human gastric carcinoma cell MGC-803 and murine cervical carcinoma cell U14 were transfected with the plasmid pEGFP-N1 and the efficiency of transfection was assessed 24 h later. Limited dilution was employed to screen and establish monoclonal cell strains, MGC-803-GFP and U14-GFP. The two fluorescent tumor cell stains were transplanted into BALB/c-nu mice and C57BL/6J mice respectively. The latency period of tumor mass appearance and the growth curve in vivo were documented. The tumor growth and metastasis were evaluated in vivo by the Viviperception Fluorescence Imagining System (VFIS). Expressions of CD44 and E-cadherin in tumor tissue were monitored by immunohistochemistry.</p><p><b>RESULTS</b>The efficiency of pEGFP-N1 transfection of MGC-803 cells and U14 cells were 30% and 60%, respectively. Monoclonal GFP(+) cell strains-MGC-803-GFP and U14-GFP were established. The latency periods of tumor formation of MGC-803-GFP and U14-GFP were 3-5 days and 2-4 days, respectively. Their tumorigenicity rates were 100% in both. The tumor growth of MGC-803-GFP was well defined by the VFIS. Only one mouse was shown to harbor lymphatic metastasis by VFIS, 60 days after transplantation. The metastasis process of U14-GFP was depicted through VFIS on 27, 37 and 52 days post-transplantation. The incidence of pulmonary metastasis and lymphatic metastasis of U14-GFP was 67% and 100% respectively when the tumor volume was >or=5 cm3. CD44 was positive and E-cadherin was negative in both tumors by immunohistochemistry.</p><p><b>CONCLUSIONS</b>Successfully established two monoclonal tumor cell strains stably expressing GFP: MGC-803-GFP and U14-GFP. Transplantation of these cells into mice can establish tumor metastasis models which could be used for future visualized tumor research in vivo.</p>


Sujets)
Animaux , Femelle , Humains , Mâle , Souris , Lignée cellulaire tumorale , Modèles animaux de maladie humaine , Protéines à fluorescence verte , Génétique , Métabolisme , Antigènes CD44 , Métabolisme , Tumeurs du poumon , Métabolisme , Anatomopathologie , Métastase lymphatique , Souris de lignée BALB C , Souris de lignée C57BL , Souris nude , Transplantation tumorale , Plasmides , Tumeurs de l'estomac , Métabolisme , Anatomopathologie , Transfection , Charge tumorale , Tumeurs du col de l'utérus , Métabolisme , Anatomopathologie
7.
Chinese Journal of Pathology ; (12): 537-541, 2009.
Article Dans Chinois | WPRIM | ID: wpr-249088

Résumé

<p><b>OBJECTIVE</b>To elucidate the expression and function of VAP-33 gene in dendritic cell sarcoma (DCS) cell line.</p><p><b>METHODS</b>The expression of VAP-33 in DCS cells was investigated by mass spectrum with immunoprecipitation membrane protein. DCS cells were treated with antigens in different dosages (150, 850, and 1500 microl) for 24, 48 and 72 h respectively. Cell morphology and phagocytosis activity of DCS cells were measured. Indirect immunofluorescence, confocal microscopy and Western blotting were used to study the distribution and expression changes of VAP-33. Moreover, DCS cells were treated with 0.5 mol/L insulin for 20 min first and followed by Western blotting to detect changes of VAP-33 and glucose transfer protein 4 (GLUT-4) in the total cellular protein, cytoplasmic protein and membrane protein. Confocal microscopy was used to document the expression and distribution changes of VAP-33 and GLUT-4 in DCS cells.</p><p><b>RESULTS</b>VAP-33 expression was obtained at the cell membrane and in the cytoplasm of DCS cells. Upon antigen stimulation, DCS cells showed more active phagocytosis and morphologically became more elongated with branched protrusions. The expression of VAP-33 was decreased by the antigen stimulation. Upon the insulin stimulation, the expression of VAP-33 and GLUT-4 were increased and co-localized.</p><p><b>CONCLUSIONS</b>VAP-33 expression in DCS originated from the dendritic cells (DC) seemed relating to the vesicle transportation during antigen processing in DC. Additionally, VAP-33 and GLUT-4 also take part in the glucose transportation in the cells.</p>


Sujets)
Animaux , Souris , Présentation d'antigène , Protéines de transport , Métabolisme , Lignée cellulaire tumorale , Membrane cellulaire , Métabolisme , Cytoplasme , Métabolisme , Sarcome à cellules dendritiques interdigitées , Métabolisme , Anatomopathologie , Régulation négative , Transporteur de glucose de type 4 , Métabolisme , Insuline , Pharmacologie , Protéines membranaires , Métabolisme , Phagocytose , Allergie et immunologie
8.
Chinese Journal of Pathology ; (12): 316-322, 2008.
Article Dans Chinois | WPRIM | ID: wpr-306022

Résumé

<p><b>OBJECTIVE</b>To investigate the effect of down-expression of inhibitor of differentiation-1 (Id-1) on the differentiation of dendritic cell sarcoma (DCS) cells in vitro.</p><p><b>METHODS</b>Down-regulation of the expression of Id-1 in DCS cells was performed by RNAi, and confirmed by protein and mRNA quantitative analyses. Cellular differentiation and biological behavior including malignant phenotypes of the cells were evaluated. All experiments included negative (no treatment group and no-target siRNA) and positive (induction-differentiation drug sodium butyrate) controls.</p><p><b>RESULTS</b>When the expression of Id-1 was down regulated, the DCS cells showed more mature morphology including cell enlargement, longer cellular extensions, more branches, and decreased nuclear/plasma ratio. Differentiation marker expression (Id-2 and CD86) was also increased. RNAi treated cells at 24 and 48 hours, showed increase percentage of cells at G0/G1 phase and less cells at S phase (P < 0.01). Importantly, the abilities of cell proliferation, colony formation and invasiveness were significantly decreased (P < 0.01), as evidenced by MTT, colony formation and transwell assays respectively.</p><p><b>CONCLUSION</b>RNAi inhibition of Id-1 protein can induce differentiation of malignant solid tumor cells along with reversion of their malignant phenotype.</p>


Sujets)
Animaux , Souris , Différenciation cellulaire , Physiologie , Prolifération cellulaire , Cellules dendritiques , Biologie cellulaire , Régulation négative , Protéines d'inhibition de la différenciation , Pharmacologie , Cellules cancéreuses en culture
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