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1.
J Lipid Res ; 55(7): 1279-87, 2014 07.
Artigo em Inglês | MEDLINE | ID: mdl-24831470

RESUMO

Cell death-inducing DFF45-like effector b (Cideb), an endoplasmic reticulum (ER)- and lipid droplet (LD)-associated protein, has been shown to play a critical role in maintaining hepatic lipid homeostasis by promoting the lipidation and maturation of VLDL particles. Here, we observed that Cideb is expressed in the jejunum and ileum sections of the small intestine, and its expression was induced by high-fat diet. Intragastric gavage with lipids resulted in the retention of lipids in the intestine in Cideb-deficient mice. In addition, we observed that mice with Cideb deficiency exhibited reduced intestinal chylomicron-TG secretion and increased lipid accumulation in the enterocytes. The sizes of chylomicrons secreted from the small intestine of Cideb-deficient mice were also smaller than those from wild-type mice. Furthermore, the overexpression of Cideb increased TG secretion and reduced lipid accumulation in the enterocyte-like Caco-2 cells. In addition, we proved that Cideb was localized to the ER and LDs and could interact with ApoB48 in Caco-2 cells. Overall, these data revealed that Cideb plays an important role in controlling intestinal chylomicron lipidation.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Quilomícrons/metabolismo , Intestino Delgado/metabolismo , Lipoilação/fisiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Células CACO-2 , Quilomícrons/genética , Humanos , Camundongos , Camundongos Mutantes
2.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 28(9): 911-4, 2012 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-22980652

RESUMO

AIM: To investigate the effects of resveratrol on the morphology of lipid droplet (LD) and the expression of lipid droplet-associated proteins in primary hepatocytes of mice. METHODS: We isolated and cultured the primary hepatocytes of mice using collagenase perfusion. The primary hepatocytes were stimulated with 200 µmol/L oleic acid (OA) for 12 h, and then added with 0 (control), 20, 50, 100 µmol/L resveratrol, respectively. Another 12 h later, we utilized Bodipy 493/503 staining to observe the morphology and amount of intracellular lipid droplets under fluorescence microscope. The Folch method and triglyceride (TG) quantitative kit were used to extract the total intracellular lipids and determine the TG contents. Western blotting was applied to analyze the expression levels of perilipin, adipophilin and TIP-47. RESULTS: Compared with the control, the size and amount of lipid droplets in primary hepatocytes were reduced after treated with different concentrations of resveratrol. The quantitative analysis showed that the intracellular TG contents decreased markedly in a dose-dependent manner, but the most significant decrease was in 50 µmol/L resveratrol group. Western blotting showed that resveratrol reduced the expressions of perilipin, adipophilin and TIP-47 in the primary hepatocytes, especially the perilipin. CONCLUSION: Resveratrol could inhibit intracellular lipid accumulation, which is most significant in 50 µmol/L concentration. The inhibiting effect of resveratrol may be caused by mediating the expression levels of lipid droplet-associated proteins in primary hepatocytes of mice.


Assuntos
Hepatócitos/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Estilbenos/farmacologia , Animais , Proteínas de Transporte/análise , Células Cultivadas , Relação Dose-Resposta a Droga , Fígado Gorduroso/metabolismo , Hepatócitos/metabolismo , Masculino , Proteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos C57BL , Perilipina-1 , Perilipina-2 , Fosfoproteínas/análise , Resveratrol , Triglicerídeos/análise
3.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 25(11): 980-3, 2009 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-19900361

RESUMO

AIM: To prepare Nano-Liposome encapsulated MAGE3/HSP70(NL M3H) and study its character and antitumor immunity in mouse. METHODS: NL M3H was prepared by the thin film-dispersion ultrasonic. The shape and size of NL M3H were detected by electron microscope. The encapsulation rate, drug-carrying capacity, stability and the releasing character were tested by Sephedex-G100 gel filtration. The mouse was immunized by NL M3H, and the antitumor immunity was detected by ELISPOT and LDH release assay. RESULTS: The mean size of NL M3H was lower than 100 nm. The encapsulation rate was 38%.The drug content was 0.038 g/L. NL M3H has good stability after stored in 4 degrees C for 6 months. The releasing profile showed that 74 percent of proteins was released during the first 24 hours in saline. The results of ELISPOT and LDH release assay showed that NL M3H generated tumor specific cytotoxic T lymphocyte(CTL)to damage tumor cell. CONCLUSION: NL M3H has novel characters, it can generate specific CTL to kill tumor cell, and can be used as new kind of vaccine against tumor.


Assuntos
Antígenos de Neoplasias/sangue , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/sangue , Vacinas Anticâncer/imunologia , Lipossomos , Nanoestruturas , Animais , Antígenos de Neoplasias/metabolismo , Vacinas Anticâncer/metabolismo , Vacinas Anticâncer/farmacocinética , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Feminino , Proteínas de Choque Térmico HSP70/metabolismo , Interferon gama/metabolismo , Lipossomos/química , Camundongos , Microscopia Eletrônica , Nanoestruturas/química , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/ultraestrutura , Fatores de Tempo
4.
Oncol Rep ; 22(4): 915-20, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19724873

RESUMO

Our previous study showed that nanoemulsion-encapsulated MAGE1-HSP70/SEA (MHS) complex protein vaccine elicited MAGE-1 specific immune response and antitumor effects against MAGE-1-expressing tumor and nanoemulsion is a useful vehicle with possible important implications for cancer biotherapy. The purpose of this study was to compare the immune responses induced by nanoemulsion-encapsulated MAGE1-HSP70 and SEA as NE(MHS) vaccine following different administration routes and to find out the new and effective immune routes. Nanoemulsion vaccine was prepared using magnetic ultrasound methods. C57BL/6 mice were immunized with NE(MHS) via po., i.v., s.c. or i.p., besides mice s.c. injected with PBS or NE(-) as control. The cellular immunocompetence was detected by ELISpot assay and LDH release assay. The therapeutic and tumor challenge assay were also examined. The results showed that the immune responses against MAGE-1 expressing murine tumors elicited by NE(MHS) via 4 different routes were approximately similar and were all stronger than that elicited by PBS or NE(-), suggesting that this novel nanoemulsion carrier can exert potent antitumor immunity against antigens encapsulated in it. Especially, the present results indicated that nanoemulsion vaccine adapted to administration via different routes including peroral, and may have broader applications in the future.


Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Sistemas de Liberação de Medicamentos/métodos , Enterotoxinas/imunologia , Proteínas de Choque Térmico HSP70/imunologia , Animais , Antígenos de Neoplasias/administração & dosagem , Vacinas Anticâncer/administração & dosagem , Citotoxicidade Imunológica/efeitos dos fármacos , Emulsões , Enterotoxinas/administração & dosagem , Ensaio de Imunoadsorção Enzimática , Proteínas de Choque Térmico HSP70/administração & dosagem , Interferon gama/efeitos dos fármacos , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Nanotecnologia/métodos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia
5.
Chin Med J (Engl) ; 122(7): 839-43, 2009 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-19493400

RESUMO

BACKGROUND: Bone morphogenetic protein (BMP) is a member of the superfamily of transforming growth factor-beta. Recent studies show that it is an indispensable factor in hematopoiesis. To better characterize the effect of recombinant human BMP (rhBMP)-2 in hematopoiesis, we set out to determine whether rhBMP-2 could promote the proliferation of mesenchymal stem cells (MSCs) and increase the levels of hematopoietic cytokines in MSCs. METHODS: 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino) carbonyl)-2H-tetrazolium hydroxide (XTT), real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the effects of rhBMP-2 on the proliferation and hematopoietic cytokine levels of MSCs. In addition, MSCs marked with Hoechst33342 were transplanted into BALB/c mice by the intravenous route or intra-bone marrow transplantation, and cluster numbers were counted. RESULTS: The XTT test revealed that rhBMP-2 significantly induced proliferation of MSCs in doses ranging from 10 ng/ml to 0.1 mg/ml in a dose-dependent manner. The experiments in vivo showed that there were more clusters of donor cells in bone marrow, spleen, liver and lung of the BMP group than those in the control group after both intra-bone marrow transplantation (P < 0.001, P < 0.001, P < 0.001, and P = 0.001, respectively) and intravenous transplantation (P < 0.001, P < 0.001, and P < 0.001 respectively). The results of real-time PCR and ELISA revealed that rhBMP-2 significantly increased mRNA expressions and protein levels of IL-6, IL-7, IL-11, G-CSF, M-CSF and SCF. CONCLUSIONS: The treatment with rhBMP-2 promotes the proliferation of MSCs in vivo and in vitro and increases the levels of hematopoietic cytokines in MSCs, which may contribute to the improvement of hematopoietic function.


Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Proliferação de Células/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Proteína Morfogenética Óssea 2 , Células Cultivadas , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Fator Estimulador de Colônias de Granulócitos/genética , Humanos , Interleucina-11/genética , Interleucina-6/genética , Interleucina-7/genética , Fator Estimulador de Colônias de Macrófagos/genética , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Fator de Células-Tronco/genética
6.
Cancer Immunol Immunother ; 58(2): 201-8, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18523770

RESUMO

Previous studies have shown that there are profuse lymphatic tissues under the intestinal mucous membrane. Moreover, vaccine administered orally can elicit both mucous membrane and system immune response simultaneously, accordingly induce tumor-specific cytotoxic T lymphocyte. As a result, the oral route is constituted the preferred immune route for vaccine delivery theoretically. However, numerous vaccines especially protein/peptide vaccines remain poorly available when administered by this route. Nanoemulsion has been shown as a useful vehicle can be developed to enhance the antitumor immune response against antigens encapsulated in it and it is good for the different administration routes. Of particular interest is whether the protein vaccine following peroral route using nanoemulsion as delivery carrier can induce the same, so much as stronger antitumor immune response to following conventional ways such as subcutaneous (sc.) or not. Hence, in the present study, we encapsulated the MAGE1-HSP70 and SEA complex protein in nanoemulsion as nanovaccine NE (MHS) using magnetic ultrasound method. We then immuned C57BL/6 mice with NE (MHS), MHS alone or NE (-) via po. or sc. route and detected the cellular immunocompetence by using ELISpot assay and LDH release assay. The therapeutic and tumor challenge assay were examined then. The results showed that compared with vaccination with MHS or NE (-), the cellular immune responses against MAGE-1 could be elicited fiercely by vaccination with NE (MHS) nanoemulsion. Furthermore, encapsulating MHS in nanoemulsion could delay tumor growth and defer tumor occurrence of mice challenged with B16-MAGE-1 tumor cells. Especially, the peroral administration of NE (MHS) could induce approximately similar antitumor immune responses to the sc. administration, but the MHS unencapsulated with nanoemulsion via po. could induce significantly weaker antitumor immune responses than that via sc., suggesting nanoemulsion as a promising carrier can exert potent antitumor immunity against antigen encapsulated in it and make the tumor protein vaccine immunizing via po. route feasible and effective. It may have a broad application in tumor protein vaccine.


Assuntos
Antígenos de Neoplasias/farmacologia , Vacinas Anticâncer/farmacologia , Proteínas de Choque Térmico HSP70/farmacologia , Nanopartículas , Proteínas de Neoplasias/farmacologia , Administração Oral , Animais , Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/toxicidade , Linhagem Celular Tumoral , Vias de Administração de Medicamentos , Emulsões/administração & dosagem , Emulsões/farmacologia , Proteínas de Choque Térmico HSP70/administração & dosagem , Proteínas de Choque Térmico HSP70/imunologia , Ativação Linfocitária , Antígenos Específicos de Melanoma , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/ultraestrutura , Proteínas de Neoplasias/administração & dosagem , Proteínas de Neoplasias/imunologia , Linfócitos T/imunologia
7.
Oncol Rep ; 20(1): 245-52, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18575744

RESUMO

Tumor antigen-derived peptides have been widely used to elicit tumor-specific cytotoxic T lymphocytes (CTLs). MAGE gene products are of particular interest owing to their wide expression in many tumors and their potential to induce tumor-specific CTL responses. Antigen-specific CTLs induced by MAGE gene-derived peptides have proven to be highly efficacious in the prevention and treatment of various types of tumors. MAGE-3 has been used as a target for tumor immunotherapy. MAGE-n is a new member of the MAGE gene family and has been shown to be closely associated with hepatocellular carcinoma (HCC). However, the majority of previous investigations focused on the single MAGE antigen-derived peptides as a cancer vaccine which has many limitations. The tumor antigen expression is known to be heterogeneous and tumor cells can express multiple tumor antigens. Thus, vaccines incorporating single antigen-derived epitopes may be inadequate in generating a complete immune response against the tumor. Instead, a polyvalent vaccine incorporating epitopes derived from several tumor antigens may be more effective. Our study combined the MAGE-3 and MAGE-n-derived peptides as a cancer vaccine. The results showed that the combination of MAGE-3 and MAGE-n epitopes induced more effective anti-tumor immune responses than either of the peptides alone. In addition, the peptide-specific activity was observed to be in an MHC-restricted manner. Our study indicated that the combination of several tumor antigen-derived peptides may present a better peptide-based cancer immunotherapy.


Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Epitopos de Linfócito T , Proteínas de Neoplasias/imunologia , Neoplasias/terapia , Linfócitos T Citotóxicos/imunologia , Antígenos de Neoplasias/genética , Linhagem Celular Tumoral , Antígeno HLA-A2/imunologia , Humanos , Interferon gama/biossíntese , Proteínas de Neoplasias/genética , Neoplasias/imunologia
8.
Cancer Biol Ther ; 7(8): 1280-7, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18497573

RESUMO

Hepatocellular carcinoma (HCC) has been reported to be resistant to Fas-mediated apoptosis. In present study, experiments were conducted to investigate the potential effects of CYP2E1 overexpression on susceptibility of HCC to Fas-mediated cytotoxicity. HCC cell line HepG2 was infected with Ad-CYP2E1 to enhance the expression of CYP2E1, followed by treatment with low toxic dose of recombinant human Fas ligand (FasL, 0.5 ng/ml) in the presence of Actinomycin D (Act D, 125 ng/ml). High level of Fas expression was found in HepG2 cells. Its protein level and distribution kept unchanged after different treatments. Compared with control, CYP2E1 expressed HepG2 cells were more sensitive to FasL plus Act D. The sensitivity was elevated in a multiplicity of infection (m.o.i)-dependent manner, which was dramatically suppressed by CYP2E1 inhibitor diallyl disulfide (DAS) (p < 0.01). The percentage of apoptotic cells caused by FasL/Act D was increased from 18.7 to 75% after infection with Ad-CYP2E1 (p < 0.01). DAS treatment resulted in 60% reduction of apoptotic ratio (p < 0.01). Antioxidants GSH ethyl ester, Vitamin C and Vitamin E efficiently protected against cytotoxicity induced by FasL plus Act D in CYP2E1-expressed HepG2 cells. After adding FasL/Act D, increased caspases activities, lipid preoxidation and reduced GSH level, as well as mitochondrial release of cytochrome c were found in Ad-CYP2E1 infected cells (all p < 0.01); these changes were significantly attenuated by DAS (all p < 0.05). These results suggested that CYP2E1 potentiates Fas-mediated HepG2 cells toxicity via the induction of oxidative stress to promote apoptosis. Adenovirus-mediated overexpresson of CYP2E1 may have an important role in the elimination of hepatoma cells mediated by immune effector cells in the liver.


Assuntos
Carcinoma Hepatocelular/patologia , Citocromo P-450 CYP2E1/metabolismo , Proteína Ligante Fas/farmacologia , Neoplasias Hepáticas/patologia , Receptor fas/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Citocromo P-450 CYP2E1/biossíntese , Citocromo P-450 CYP2E1/genética , Proteína Ligante Fas/genética , Humanos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Receptor fas/genética
9.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 24(5): 457-60, 2008 May.
Artigo em Chinês | MEDLINE | ID: mdl-18466701

RESUMO

AIM: To prepare nanoemulsion-encapsulated MAGE1-Hsp70/SEA and to evaluate its anti-tumor effects in mouse. METHODS: Nanoemulsion vaccine NE(MHS) was prepared using magnetic ultrasound methods and used to immunize C57BL/6 mice. The cellular immune responses were detected by IFN-gamma ELISPOT and LDH release assay. The tumor challenge assay was performed too. RESULTS: (1) The mean size of NE(MHS) was (20+/-5) nm. The encapsulation rate was 87% and the nanoemulsion vaccine had a good stability. (2) The frequency of MAGE-1 specific CTL and cytotoxicity of CTL to B16-MAGE-1 cells were both greatly enhanced in immunization group than those in control group (P<0.05). NE(MHS) could significantly delay the appearance of tumors and increase the percentage of tumor-free mice. CONCLUSION: The nanoemulsion had excellent physical and chemical characteristics. It could elicit MAGE-1-specific cellular immune response and anti-tumor effects against the MAGE-1-expressing tumor.


Assuntos
Antígenos de Neoplasias/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Proteínas de Choque Térmico HSP70/administração & dosagem , Interferon gama/uso terapêutico , Ativação Linfocitária/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Proteínas de Neoplasias/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Animais , Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Vacinas Anticâncer/administração & dosagem , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Imunização , Interferon gama/administração & dosagem , Ativação Linfocitária/imunologia , Masculino , Melanoma Experimental/imunologia , Antígenos Específicos de Melanoma , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T Citotóxicos
10.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 24(3): 278-81, 2008 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-18328193

RESUMO

AIM: To observe the effects of hepatoma-targeting recombinant adenovirus vectors of staphylococcal enterotoxin A (SEA) and/or CD80 gene on hepatoma and to study its immunological mechanisms. METHODS: Using AdEasy adenovirus system, we constructed recombinant adenovirus vectors of SEA and/or CD80 gene driven by alpha-fetoprotein (AFP) enhancer I and promoter. After intratumoral therapy for the mice bearing subcutaneous xenograft hepatoma with the recombinant adenoviruses, SEA and/or CD80 mRNA and protein were detected by RT-PCR and Western blot. IFN-gamma-producing cell frequency and specific cytotoxicity of T lymphocytes to Hepa1-6 cells were detected by ELISpot and LDH-released assay in the splenocytes. Effects of recombinant adenoviruses on hepatoma were assessed by changes of tumor volumes and survival time in the treated mice. RESULTS: The recombinant adenoviruses constructed by us made SEA and/or CD80 mRNA and protein targetedly express in hepatoma tissues. When compared with the empty vector and PBS groups, the IFN-gamma-producing cell frequency and specific cytotoxicity of T lymphocytes increased, the tumor volumes of mice decreased and the survival time increased in the double-gene and single-gene groups. Double genes elicited better antitumor effects and stronger immune responses. There were no significant differences in the effects between CD80 group and SEA group or between empty vector group and PBS group. CONCLUSION: The hepatoma-targeting recombinant adenovirus vectors constructed in this study can elicit effective antitumor effects on hepatoma and the effects of double genes are better than that of single gene.


Assuntos
Adenoviridae/genética , Carcinoma Hepatocelular/terapia , Enterotoxinas/fisiologia , Terapia Genética/métodos , Neoplasias Hepáticas/terapia , Animais , Antígeno B7-1/genética , Antígeno B7-1/metabolismo , Western Blotting , Elementos Facilitadores Genéticos/genética , Enterotoxinas/genética , Enterotoxinas/metabolismo , Feminino , Vetores Genéticos/genética , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , alfa-Fetoproteínas/genética
11.
Viral Immunol ; 20(3): 421-8, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17931112

RESUMO

Although Kikuchi-Fujimoto disease (KFD) has a higher prevalence among Asian countries, it is a well-defined entity throughout the world. However, its etiology and pathogenesis remain undetermined. To study whether B19 infection is associated with idiopathic KFD (iKFD), we examined the presence of the viral genome and proteins in paraffin-embedded tissues of lymph nodes retrospectively from 33 iKFD patients and 16 age- and sex-matched control subjects by nested PCR (nPCR), in situ hybridization (ISH), and immunohistochemistry (IHC). B19 was detected in 87.1, 69.7, and 57.6% of iKFD specimens by nPCR, ISH, and IHC, respectively, whereas the virus was positive in only 56.3, 31.3, and 25.0% of control tissues by the respective methods (nPCR: p = 0.029; ISH: p = 0.011; IHC: p = 0.032). The IHC-ISH double-staining assay demonstrated that B19-infected cells were mainly lymphocytes and a small number of histiocytes. These results showed for the first time a high frequency of localized persistence of B19 in lymph nodes from iKFD patients, suggesting that B19 might play an important role in the pathogenesis of iKFD.


Assuntos
Linfadenite Histiocítica Necrosante/etiologia , Linfadenite Histiocítica Necrosante/virologia , Infecções por Parvoviridae/virologia , Parvovirus B19 Humano , Adolescente , Adulto , Idoso , Criança , DNA Viral/análise , Feminino , Histiócitos/virologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Linfonodos/virologia , Linfócitos/virologia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Estudos Retrospectivos
12.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 22(5): 613-6, 2006 Sep.
Artigo em Chinês | MEDLINE | ID: mdl-16948908

RESUMO

AIM: To construct hepatoma-targeting recombinant co-expression adenovirus vector of Staphylococcal enterotoxin A (SEA) and CD80 gene. METHODS: Us-ing the adenovirus transfer plasmids pShuttle and pShuttle-CMV, we constructed a new transfer plasmid pShuttle2 with polyA signal sequence instead of CMV enhancer/promoter. AFP enhancer, promoter, SEA or CD80 gene was subcloned into pShuttle2 from the vectors pKS-EP or pMD18-T-BIS respectively, and then the constructed plasmid pShuttle2-BIS containing AFP enhancer, promoter, SEA or CD80 gene was cotransformed into E.coli BJ5183 with backbone vector pAdEasy-1 to obtain recombinant adenovirus DNA. The recombinant adenovirus DNA was transfected into 293 cells to prepare adenovirus. After AFP-producing cell line Hepa1-6 and AFP-nonproducing cell lines B16 and NIH3T3 were infected by recombinant adenovirus, the expression of SEA and CD80 on the surface of cells was detected by indirect immunofluorescent staining, laser confocal microscope and flow cytometry (FCM). RESULTS: SEA and CD80 was specifically co-expressed on the surface of infected Hepa1-6 cells but not on B16 and NIH3T3 cells. CONCLUSION: Hepatoma-targeting recombinant co-expression adenovirus vector of SEA and CD80 gene was successfully constructed, which lays the foundation for further research on application of SEA and CD80 in targeted gene therapy for hepatoma and the underlying immunological mechanisms.


Assuntos
Adenoviridae/genética , Antígeno B7-1/genética , Carcinoma Hepatocelular/genética , DNA Recombinante/genética , Enterotoxinas/genética , Vetores Genéticos/genética , Staphylococcus , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/terapia , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Enzimas de Restrição do DNA/metabolismo , DNA Viral/genética , Feminino , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica , Terapia Genética , Vetores Genéticos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/genética , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase
13.
Biochem Biophys Res Commun ; 347(1): 208-14, 2006 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-16814254

RESUMO

Staphylococcus enterotoxin A (SEA) stimulates T cells bearing certain TCR beta-chain variable regions, when bound to MHC-II molecules, and is a potent inducer of CTL activity and cytokines production. To decrease toxicity of SEA to the normal MHC-II(+) cells and to localize the immune response induced by SEA to the tumor site, my colleague previously genetically fused SEA with B7.1 transmembrane region (named as SEAtm) to make SEA express on the surface of tumor cells and tumor cells modified with SEAtm could induce efficient antitumor immunity in vitro. The tumor cell vaccines modified with multiple immune activators frequently elicited stronger antitumor immune responses than single-modified vaccines. In this study, we modified the tumor cell vaccine with B7.1 and SEAtm to improve efficiency in the application of SEA. First, SEAtm gene was subcloned from recombinant plasmid pLXSNSEP by PCR and murine B7.1 gene was cloned from splenocytes derived from C57BL/6 mice by RT-PCR. Then, the eukaryotic co-expression vector of SEA and murine B7.1 gene was constructed and named as pcDNA-BIS. B16 cell lines stably expressing SEA and/or B7.1 were established by screening with G418 after transfection and inactivated for the preparation of tumor cell vaccines to treat mice bearing established B16 tumors. The results indicated that the dual-modified tumor cell vaccine B16/B7.1+SEAtm (B16-BIS) elicited significantly stronger antitumor immune responses in vivo when compared with the single-modified tumor cell vaccines B16/B7.1 (B16-B7.1) and B16/SEAtm (B16-SEAtm), and supported the feasibility and effectiveness of the dual-modified tumor cell vaccine with superantigen and co-stimulatory molecule.


Assuntos
Antígeno B7-1/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Enterotoxinas/imunologia , Melanoma/imunologia , Melanoma/prevenção & controle , Animais , Antígeno B7-1/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Enterotoxinas/genética , Feminino , Imunidade Inata/imunologia , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Resultado do Tratamento
14.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 22(4): 450-1, 453, 2006 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-16806006

RESUMO

AIM: To construct the eukaryotic expression vector of tumor antigen MAGE-3 and establish human hepatocellular carcinoma cell line (HHCC) expressing MAGE-3. METHODS: The MAGE-3 gene was amplified by PCR and cloned into the eukaryotic expression vector pIRES2-EGFP to construct the pIRES2-EGFP-MAGE-3 plasmid. The recombinant plasmid pIRES2-EGFP-MAGE-3 was transfected into HHCC cells by lipofectamine, and then the positive clones were screened by G418. The expression of enhanced green fluorescent protein (EGFP) and MAGE-3 mRNA in positive clones were detected by fluorescence microscope and RT-PCR, respectively. RESULTS: The eukaryotic expression vector pIRES2-EGFP-MAGE-3 was successfully constructed. The expression of EGFP was found by fluorescence microscope detection and MAGE-3 mRNA transcription was detected by RT-PCR in the positive clones. CONCLUSION: The stable MAGE-3-transfected HHCC cell line is successfully established, which will provide experimental basis for further study on immunotherapy for hepatocellular carcinoma using MAGE-3 as target antigen.


Assuntos
Antígenos de Neoplasias/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proteínas de Neoplasias/genética , Animais , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/terapia , Linhagem Celular Tumoral , Enzimas de Restrição do DNA/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transfecção
15.
Cancer Immunol Immunother ; 55(7): 841-9, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16155756

Assuntos
Formação de Anticorpos/efeitos dos fármacos , Antígenos de Neoplasias/farmacologia , Vacinas Anticâncer/farmacologia , Proteínas de Choque Térmico HSP70/farmacologia , Imunidade Celular/efeitos dos fármacos , Melanoma Experimental/terapia , Nanoestruturas , Proteínas de Neoplasias/farmacologia , Animais , Anticorpos Antineoplásicos/biossíntese , Anticorpos Antineoplásicos/imunologia , Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Citotoxicidade Imunológica/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Emulsões , Ensaio de Imunoadsorção Enzimática , Proteínas de Choque Térmico HSP70/administração & dosagem , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/imunologia , Melanoma Experimental/imunologia , Antígenos Específicos de Melanoma , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/administração & dosagem , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Veículos Farmacêuticos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Organismos Livres de Patógenos Específicos , Vacinação , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/farmacologia , Vacinas Sintéticas/uso terapêutico
16.
Cancer ; 104(12): 2775-83, 2005 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-16284993

RESUMO

BACKGROUND: An unbalance of cell proliferation and cell apoptosis is an important mechanism in carcinogenesis, and angiogenesis also plays a crucial role in tumorigenesis. Recently, survivin has been identified as an important member of the inhibitor of apoptosis protein (IAP) family. Although it has been shown that survivin is highly expressed in gliomas, and is associated with tumorigenesis, progression, and poor prognosis of gliomas, as yet the relation of survivin expression with proliferation, apoptosis, and angiogenesis of gliomas it is still unclear. METHODS: Eighty-three cases of brain glioma were chosen and protein expressions of survivin and proliferating cell nuclear antigen (PCNA) in glioma cells and Factor VIII-related antigen (FVIII-RAg) in vascular endothelial cells were investigated by immunohistochemistry. Apoptotic cells of brain glioma were screened by TdT-mediated dUTP nick end-labeling (TUNEL), and survivin immunoreactivity score (IRS), proliferative index (PI), apoptotic index (AI), overall daily growth (ODG), and microvessel density (MVD) in brain gliomas were measured. RESULTS: The survivin IRS, PI, AI, ODG, and MVD of brain gliomas were 3.75 +/- 3.89, 28.39 +/- 19.49%, 1.00 +/- 0.80%, 12.19 +/- 10.21%, and 62.75 +/- 31.50, respectively, and all of them increased markedly with an increase in the pathologic grade of brain gliomas (P < 0.001 for all). PI, ODG, and MVD in the survivin-positive group were significantly higher than those in the survivin-negative group (P < 0.001 for all). PI, ODG, and MVD were positively correlated with survivin IRS (P < 0.001 for all). Although there was no significant difference between AI in the survivin-positive group or in the survivin-negative group (P = 0.108), AI was inversely correlated with survivin IRS (P = 0.005). CONCLUSIONS: Survivin is overexpressed in brain gliomas, which may play an important role in malignant proliferation, antiapoptosis, and angiogenesis of brain gliomas.


Assuntos
Neoplasias Encefálicas/patologia , Transformação Celular Neoplásica/patologia , Glioma/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Adulto , Idoso , Apoptose/fisiologia , Biomarcadores Tumorais/análise , Biópsia por Agulha , Neoplasias Encefálicas/fisiopatologia , Proliferação de Células , Estudos de Coortes , Progressão da Doença , Feminino , Glioma/fisiopatologia , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Proteínas Inibidoras de Apoptose , Masculino , Proteínas Associadas aos Microtúbulos/análise , Pessoa de Meia-Idade , Proteínas de Neoplasias/análise , Neovascularização Patológica , Valor Preditivo dos Testes , Estudos de Amostragem , Sensibilidade e Especificidade , Survivina
17.
Zhonghua Wai Ke Za Zhi ; 43(13): 885-8, 2005 Jul 01.
Artigo em Chinês | MEDLINE | ID: mdl-16083611

RESUMO

OBJECTIVE: To investigate the expression level of inhibitor of apoptosis protein survivin gene in human brain glioma and its role in malignant proliferation and antiapoptosis of brain glioma. METHODS: Eighty-three cases of brain glioma specimen was chosen, protein expression of survivin and proliferating cell nuclear antigen (PCNA) was investigated by immunohistochemistry streptavidin-biotin complex (SABC) method, the immunoreactivity score (IRS) of survivin and the proliferative index (PI) were counted. Apoptotic cells were screened by TdT-mediated dUTP-biotin nick-end labeling (TUNEL) method, and the apoptotic index (AI) of brain glioma was calculated. RESULTS: The survivin IRS, PI and AI of brain glioma were 3.8 +/- 3.9, (28.4 +/- 19.5)% and (1.0 +/- 0.8)% respectively, and all of them were elevated with the increase of pathological grade of brain glioma (P < 0.01 for all). PI in survivin positive group was significantly higher than that in survivin negative group (P < 0.01), and PI was positively correlated with survivin IRS (r = 0.740, P < 0.01). There was no significant difference between AI in survivin positive group and that in survivin negative group (P > 0.05), however, AI was negatively correlated with survivin IRS (r = -0.307, P < 0.01). CONCLUSIONS: Survivin is overexpressed in brain glioma, and which may play important roles in malignant proliferation and antiapoptosis of brain glioma.


Assuntos
Apoptose , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proliferação de Células , Glioma/genética , Glioma/patologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , Adolescente , Adulto , Idoso , Neoplasias Encefálicas/metabolismo , Criança , Pré-Escolar , Feminino , Glioma/metabolismo , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Proteínas Inibidoras de Apoptose , Masculino , Proteínas Associadas aos Microtúbulos/biossíntese , Pessoa de Meia-Idade , Proteínas de Neoplasias/biossíntese , Antígeno Nuclear de Célula em Proliferação/biossíntese , Survivina
18.
Space Med Med Eng (Beijing) ; 16(4): 248-52, 2003 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-14594029

RESUMO

OBJECTIVE: To investigate the metabolic changes of calcium and phosphorus in dentin, dental pulp and periodontium in tail-suspended rats, and the functions of TGF-beta 1, c-fos, collagen-I and collagen IV in dentin, dental pulp and periodontium. METHOD: Relative percentage contents of Ca, P in dentin, dental pulp and periodontium were measured with scanning electron microscope and energy spectrum analytical system in 3 groups of rats. The expression of TGF-beta 1, c-fos, collagen-I and collagen IV were also observed. RESULT: In the suspension group, the relative percentage content of Ca declined significantly, while P increased slightly. There were no significant differences of Ca, P in alveolar bone. The expressions of TGF-beta 1, c-fos and collagen-I declined, but the expression of collagen-IV in pulp vessel increased. There were no significant changes of expressions of TGF-beta 1, c-fos, collagen-I and collagen-IV in the vicinity of PDL. After adopting artificial countermeasures, the above expressions restored partly. CONCLUSION: Weightlessness might cause abnormal mineralization in dentin, and 1.5 G artificial countermeasures could eliminate the above changes of mineral metabolism. The poor mineralization of dentin might be associated with the reduced secretion of TGF-beta 1, c-fos and collagen-I in tail-suspended rats.


Assuntos
Polpa Dentária/metabolismo , Dentina/metabolismo , Elevação dos Membros Posteriores/fisiologia , Hipergravidade , Periodonto/metabolismo , Simulação de Ausência de Peso , Animais , Cálcio/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo IV/metabolismo , Polpa Dentária/ultraestrutura , Dentina/ultraestrutura , Microscopia Eletrônica de Varredura , Fósforo/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Calcificação de Dente/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Contramedidas de Ausência de Peso
19.
World J Gastroenterol ; 9(6): 1270-2, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12800238

RESUMO

AIM: To explore and discuss the clinicopathologic characteristics of mucosa-associated lymphoid tissue (MALT) lymphoma in gastroscopic biopsy specimen. METHODS: A retrospective study of 26 cases of lymphoma diagnosed by gastroscopic biopsy during 1999 to 2001 from gastroscopy files of Xijing Hospital was made. The diagnostic criteria were adopted according to the new classification of non-Hodgkin's lymphoma. RESULTS: Twenty-six cases of primary gastric lymphoma consisting of 15 men and 11 women, aged between 23 to 76 years were recruited from 6 225 cases who received gastroscopy. All of them were diagnosed by both endoscopic findings and histological examinations. Histologically, 23 cases were MALToma (low grade) and 3 cases lymphoblastic lymphoma (high grade). Immunohistochemically, all cases were CD20 positive, while CK and EMA were negative. CONCLUSION: The majority of the cases of primary low-grade gastric lymphoma have morphologic and clinical features that justify their inclusion in the category of low-grade lymphoma of mucosa associated lymphoid tissue.


Assuntos
Gastroscopia , Linfoma de Zona Marginal Tipo Células B/patologia , Adulto , Idoso , Biópsia , Feminino , Humanos , Imuno-Histoquímica , Linfoma de Zona Marginal Tipo Células B/metabolismo , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos
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