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1.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 32(2): 476-482, 2024 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-38660855

RESUMO

OBJECTIVE: To study the reversal effect of NVP-BEZ235 on doxorubicin resistance in Burkitt lymphoma RAJI cell line. METHODS: The doxorubicin-resistant cell line was induced by treating RAJI cells with a concentration gradient of doxorubicin. The levels of Pgp, p-AKT, and p-mTOR in cells were detected by Western blot. Cell viability was detected by MTT assay. IC50 was computed by SPSS. RESULTS: The doxorubicin-resistant Burkitt lymphoma cell line, RAJI/DOX, was established successfully. The expression of Pgp and the phosphorylation levels of AKT and mTOR in RAJI/DOX cell line were both higher than those in RAJI cell line. NVP-BEZ235 downregulated the phosphorylation levels of AKT and mTOR in RAJI/DOX cell line. NVP-BEZ235 inhibited the proliferation of RAJI/DOX cell line, and the effect was obvious when it was cooperated with doxorubicin. CONCLUSION: The constitutive activation of PI3K/AKT/mTOR pathway of RAJI/DOX cell line was more serious than RAJI cell line. NVP-BEZ235 reversed doxorubicin resistance of RAJI/DOX cell line by inhibiting the PI3K/AKT/mTOR signal pathway.


Assuntos
Linfoma de Burkitt , Proliferação de Células , Doxorrubicina , Resistencia a Medicamentos Antineoplásicos , Imidazóis , Proteínas Proto-Oncogênicas c-akt , Quinolinas , Serina-Treonina Quinases TOR , Humanos , Doxorrubicina/farmacologia , Linhagem Celular Tumoral , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinolinas/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Proliferação de Células/efeitos dos fármacos , Imidazóis/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Sobrevivência Celular/efeitos dos fármacos , Fosforilação
2.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(2): 433-438, 2019 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-30998150

RESUMO

OBJECTIVE: To investigate the effect of steadily down-regulating the expression of calreticulin (CALR) on the invasion of natural killer/T-cell lymphoma SNK6 cells, and explore its possible mechanism. METHODS: The sequences of specific short hairpin RNA (shRNA) targeting on human CALR were designed, and were inserted into pLKO.1-puro lentivirus vector, and the reconbinant lentivirus vector was obtained; the lentivirus particles were backed by three-plasmid system and transfected into SNK6 cells, the SNK6 cells stably down-regulating the CALR expression were sercened by puromytain, the CALR-silencing effect was verified by real-time PCR and Western blot. CCK-8 assay was used to evaluate the cell viability, The transwell invasion assays was used to analyse invasion of SNK6 cells. The mRNA expression of Calreticulin, MMP2, MMP9 and VEGF was determined by real time PCR, the protein expression of Calreticulin and GAPDH was analyzed by Western blot. RESULTS: The recombinant lentiviral vector pLKO.1-puro-shCALR was successfully constructed, packed into the lentivirus, then the SNK6 cells stably down-regulating Calreticulin expression was obtained. When Calreticulin was down-rengulated in SNK6 cells, the proliferation rate was reduced and the invasion ability was decreased; the mRNA levels of VEGF and MMP-2/9 also were reduced. CONCLUSION: The stable down-regnlation of CALR expression in SNK6 cells can attenuate the imvasiveness of SNK6 cells, which maybe related with transcriptional decrease of MMP2, MMP9 and VEGF.


Assuntos
Calreticulina/metabolismo , Calreticulina/genética , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Vetores Genéticos , Humanos , Lentivirus , Metaloproteinase 2 da Matriz , Metaloproteinase 9 da Matriz , Interferência de RNA , RNA Interferente Pequeno , Transfecção , Fator A de Crescimento do Endotélio Vascular
3.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 25(5): 1397-1405, 2017 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-29070114

RESUMO

OBJECTIVE: To explore the effects of mTOR inhibitor rapamycin on proliferation, cell cycle and apoptosis of Burkitt's lymphoma cell line Raji and CA46 cells and its mechanism, so as to provide the experimental evidence for a therapeutic target of Burkitt's lymphoma. METHODS: 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay was performed to assess the inhibitory effect of rapamycin on proliferation of Burkitt's lymphoma cell line Raji and CA46 cells. The cell cycle distribution of Raji and CA46 cells was analyzed by flow cytometry with propidium iodide(PI) single staining. The cell apoptosis of Raji and CA46 cells was analyzed by flow cytometry with FITC Annexin V+PI double staining. The expressions of RPS6, p-RPS6, survivin and caspase-3 proteins were detected by Western blot after treating with rapamycin. RESULTS: Rapamycin markedly inhibited the proliferation of both Raji and CA46 cells in a time- and concentration-dependent manners, showing good biological activity, the cell proliferation inhibition rate reached about 20% after treatment with 1 nmol/L rapamycin. After treatment with different concentrations of rapamycin for 24 and 48 hours, the proportion of both cells in G1/G0 phase in the treated groups was significantly increased in a time- and concentration-dependent manners in comparison with the solvent control group. With regard to the cells in S and G2/M phase, the decreased population was accompanied by the increase of G1/G0 phase cells. After treatment with 100 nmol/L rapamycin for 48 hours, both Raji and CA46 cells demonstrated an apparent apoptosis,especially late apoptosis by flow cytometry with Annexin V+PI staining. After treatment with rapamycin, the expression of p-RPS6 and survivin of Raji and CA46 cells was obviously down-regulated, the expression of caspase-3 was obviously up-regulated in a time- and dose-dependent manners. However, rapamycin did not obviously affect the expression of RPS6. CONCLUSION: The rapamycin can effectively inhibit cell proliferation, arrest Raji and CA46 cells in G1/G0 phase, and this effect associates with inhibiting the activation of mTOR/RPS6 signal pathway through down-regulating the expression of phosphorylated RPS6, i.e. mTOR downstream signal pathway. It also can induce apoptosis by down-regulating the expression of anti-apoptotic protein survivin and activating the intrinsic pro-apoptotic protein caspase-3.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linfoma de Burkitt/tratamento farmacológico , Sirolimo/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Serina-Treonina Quinases TOR
4.
Cell Physiol Biochem ; 33(3): 569-80, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24603109

RESUMO

BACKGROUND: Mesenchymal stem cells are capable of self-renewal and multi-lineage differentiation. They are used extensively to treat several diseases. Traditionally, mesenchymal stem cells are cultured in serum-containing media, typically supplemented with fetal bovine serum (FBS). However, the variability of FBS is likely to skew experimental results. Although serum-free media used to expand mesenchymal stem cells has facilitated remarkable achievements, immunomodulation of these cells in under serum-free conditions is poorly understood. We hypothesized that mesenchymal stem cells expanded in serum-free media will retain powerful immunoregulatory functions in vitro and in vivo. DESIGN AND METHODS: Immunosuppressive activity and the immunomodulatory cytokines produced by mesenchymal stem cells in serum-free media were characterized in vitro. Immunomodulation by serum-free mesenchymal stem cell expansion in monocrotaline-induced pulmonary hypertension was explored in vivo. RESULTS: Similar to cells in serum-containing media, mesenchymal stem cells expanded in serum-free media inhibited proliferation and apoptosis of CD4(+)T cells. They also exhibited strong immunosuppressive activities and secreted high levels of immunomodulatory cytokines such as PGE2, IDO1, COX2, IL-6, and IL-1ß, but not HGF. On the other hand, growth of mesenchymal stem cells in serum-free media attenuated pulmonary vascular remodeling and inhibited mRNA expression of proinflammatory cytokines TNF-α, IFN-γ, IL-6, IL-1ß, and IL-18. CONCLUSIONS: Mesenchymal stem cells in serum-free media maintained powerful immunomodulatory function in vitro and in vivo; serum-free media may replace serum-containing media for basic research and clinical applications.


Assuntos
Apoptose/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células/efeitos dos fármacos , Meios de Cultura Livres de Soro/farmacologia , Tolerância Imunológica/efeitos dos fármacos , Células-Tronco Mesenquimais/imunologia , Animais , Apoptose/imunologia , Bovinos , Ciclo-Oxigenase 2/imunologia , Citocinas/imunologia , Dinoprostona/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Células-Tronco Mesenquimais/citologia
5.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 17(4): 929-32, 2009 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-19698231

RESUMO

The purpose of this study was to investigate the lethal effect of cytotoxic lymphocytes against U266 cells induced by DCs pulsed with multiple myeloma (MM) U266 lysate and transfected with GM-CSF recombinant adenovirus. The cytotoxic lymphocytes against U266 cells were induced by culturing with DCs, which pulsed with MM U266 antigens and transfected with GM-CSF recombinant adenovirus. The effect of cytotoxic lymphocytes against U266 cells were measured by LDH release detection. Experiments were divided into 3 groups: N-DC group as control in which DCs were normal; U-DC group in which DCs were pulsed by U266 soluble antigen, and G-U-DC group in which DCs were stimulated by U266 soluble antigen and GM-CSF transfected with Ad-CMV. The results showed that there was significant difference on killing rate against U266 cells between 3 groups (F = 10.939, p < 0.05). The killing rate of G-U-DC group was the highest (p < 0.001), and killing rate of U-DC group was higher than that of N-DC group (p < 0.001). It is concluded that the cytotoxic lymphocytes against U266 cells can be induced by DCs pulsed with U266 lysate, and the lethal effect of CTLs can be enhanced when DCs transfected by recombinant adenovirus with exogenous gene GM-CSF.


Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Linfócitos T Citotóxicos/imunologia , Adenoviridae/genética , Antígenos de Neoplasias/genética , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Mieloma Múltiplo , Proteínas Recombinantes , Transfecção
6.
Chin Med J (Engl) ; 121(21): 2180-4, 2008 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-19080181

RESUMO

BACKGROUND: Survivin is a rather specific gene in tumor tissue. We transfected dendritic cells (DCs) with recombinant adenovirus (Ad) containing survivin gene and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene and tested the inducing effect of the transfected DCs on cytotoxic T lymphocytes (CTL) to kill leukemic cells. METHODS: After derived from the peripheral, DCs was assayed by mixed leukocyte reaction (MLR) tests. Lactate dehydrogenase (LDH) release test was used to evaluate cytotoxicity of CTL. RESULTS: Expression of survivin in transfected DCs was confirmed by Western blotting analysis. GM-CSF expression was confirmed by enzyme-linked immunosorbent assay (ELISA). In MLR assay, DCs coinfected with Ad-survivin and Ad-GM-CSF induced higher allogeneic lymphocyte reaction than control DCs at ratios of 1:5, 1:10, 1:50 and 1:100. DCs coinfected with Ad-survivin and Ad-GM-CSF had much higher activity of CTL to HL-60 cells than DCs infected with Ad-survivin only, Ad-GM-CSF only, or control DCs. Levels of interleukin-12 (IL-12) and interferon gamma (IFN-gamma) in lymphocyte supernatants containing DCs coinfected with Ad-survivin and Ad-GM-CSF were significantly higher than those in the control group. CONCLUSION: DCs coinfected with Ad-survivin and Ad-GM-CSF induce much higher anti-leukemic response in vitro than those infected with either factor. Therefore, adenovirus vectors containing survivin and GM-CSF genes may be promising vaccine candidates for leukemia therapy.


Assuntos
Células Dendríticas/fisiologia , Terapia Genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Leucemia/terapia , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , Linfócitos T Citotóxicos/imunologia , Adenoviridae/genética , Citotoxicidade Imunológica , Células Dendríticas/ultraestrutura , Células HL-60 , Humanos , Proteínas Inibidoras de Apoptose , Interferon gama/biossíntese , Interleucina-12/biossíntese , Ativação Linfocitária , Survivina , Transfecção
7.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 15(3): 591-3, 2007 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-17605872

RESUMO

This study was purposed to investigate the immunological effects of modified dendritic cells (DCs) in inducing cytotoxic T cells (CTLs) effect against lymphoma cells. The DCs were derived from human peripheral blood and transfected with recombined adenovirus vector carrying survivin gene, Western blot was used to detect the expression of survivin, the lactate dehydrogenase (LDH) release test was used to determine the cytotoxicity of CTLs, the mixed lymphocyte reaction (MLR) was used to measure the ability to proleferate allo-lymphocyte by DCs, ELISA was used to assay IL-12 level in supernatant. The results showed that the expression of survivin in transfected dentritic cells was confirmed by Western blot analysis. In MLR assay, DCs transfected with Ad-survivin could induce higher allogeneic lymphocytes reaction at the ratios of 1:5, 1:10, 1:50 and 1:100. DCs transfected with Ad-survivin had much higher activity of CTL to CA46 cells than control DCs. The levels of IL-12 of supernatants containing DCs transfected with Ad-survivin were significantly higher than that in the control group. It is concluded that DCs transfected with Ad-survivin can induce CTL response in vitro against lymphoma cells.


Assuntos
Adenoviridae/genética , Células Dendríticas/imunologia , Linfoma/imunologia , Proteínas Associadas aos Microtúbulos/genética , Linfócitos T Citotóxicos/imunologia , Adenoviridae/metabolismo , Apoptose/imunologia , Vetores Genéticos/genética , Humanos , Proteínas Inibidoras de Apoptose , Interleucina-12/metabolismo , Proteínas Associadas aos Microtúbulos/imunologia , Recombinação Genética , Survivina , Transfecção , Células Tumorais Cultivadas
8.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 14(4): 791-4, 2006 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-16928323

RESUMO

The study was aimed to construct the recombinant adenovirus vectors containing human survivin gene, and to investigate their expression in transfected dendritic cells. Full length cDNA encoding survivin was obtained by PCR amplification from plasmid pcDNA3.0-survivin. The PCR product was restricted, and then inserted into pShuttle-CMV. The plasmids of pShuttle-CMV-survivin were linearized with PmeI, and the fragment containing survivin was ligated with pShuttle-CMV and transfected into E. coli BJ5183. After homologous recombination in bacteria, the extracted plasmid from the positive bacteria were linearized with PacI, transfected into HEK293 cells with liposome Lipofectamine 2000. Then, the harvested adenovirus supernatants were transfected into dendritic cells. The results showed that the recombinant adenovirus-survivin was constructed successfully and its titer was about 2.65 x 10(9) pfu/ml. The expression of survivin in transfected dendritic cells was confirmed by Western blot analysis. It is concluded that the recombinant adenovirus vector containing human survivin was constructed successfully, which may provide preliminary laboratory evidence for anti-leukemia immunotherapy.


Assuntos
Adenoviridae/metabolismo , Células Dendríticas/metabolismo , Vetores Genéticos , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas de Neoplasias/biossíntese , Adenoviridae/genética , Células Dendríticas/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Viral da Expressão Gênica , Humanos , Proteínas Inibidoras de Apoptose , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Survivina , Transfecção
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