RESUMEN
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The reported data indicate that oleic acid (OA) decreases cholesterol absorption. To explore the underlying mechanisms, the effects of OA on the expression of cholesterol transport-related proteins (NPC1L1, ABCG5/8, ACAT2, MTP) and the unfolded protein response (UPR) pathway were studied in CaCo-2 enterocytes by incubating CaCo-2 cells with taurocholate micelles or taurocholate micelles containing different concentrations of OA (0.251.0 mM). We show that OA effectively induces XBP1 mRNA splicing, a key component of the UPR signaling, and the expression of BiP and mature ATF6 proteins in a concentration-dependent manner, leading to the induction of endoplasmic reticulum (ER) stress and activation of the UPR. Interestingly, OA decreases NPC1L1 expression in a dose-dependent manner while it has no effects on ABCG5 and MTP mRNA level or SREBP-2, ABCG8, and ACAT2 protein level. In CaCo- (..) (AU)
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Humanos , Ácidos Oléicos/farmacocinética , Proteínas de Transferencia de Ésteres de Colesterol/fisiología , Estrés del Retículo Endoplásmico/fisiología , Células CACO-2/fisiología , Respuesta de Proteína Desplegada/fisiología , Absorción Intestinal/fisiologíaRESUMEN
BACKGROUND AND AIM: Chinese traditional medical science is generally used as a therapeutic method against functional dyspepsia (FD) in China. Although great effort is made to understand the pharmaceutical mechanisms of Chinese traditional medicine, such as typical traditional Chinese medicine, Wei Kangning, there are still many mysteries to be uncovered. METHODS: The model of FD was established by stimulating rats via tail damping and the rats were treated with traditional Chinese medicine, Wei Kangning. The proteins of the rat gastrointestinal tissues were extracted and run by 2-DE, then the differential proteins were identified using matrix-assisted laser desorption ionisation time-of-flight mass spectrometry and validated with Western blotting or fluorescent quantitation polymerase chain reaction. RESULTS: A total of 228 unique proteins in FD model rats were detected with significant changes in their expression levels corresponding with traditional Chinese medicine, Wei Kangning, administration. Twenty-eight of these proteins were identified, which are involved in many biological functions, such as organism antioxidant enzymes, energy metabolism, glutathione S-transferase, pi2, superoxide dismutase 2 and alpha-enolase and so on. CONCLUSIONS: These proteomic results presented therefore provide additional support to the hypothesis that glutathione S-transferase, pi2, superoxide dismutase 2, α-enolase and voltage-dependent anion channel are the targets of FD treated with traditional Chinese medicine, Wei Kangning.
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Medicamentos Herbarios Chinos/farmacología , Dispepsia/tratamiento farmacológico , Intestinos/efectos de los fármacos , Proteómica , Estómago/efectos de los fármacos , Estrés Psicológico/complicaciones , Animales , Western Blotting , Modelos Animales de Enfermedad , Dispepsia/etiología , Dispepsia/metabolismo , Dispepsia/fisiopatología , Electroforesis en Gel Bidimensional , Mucosa Gástrica/metabolismo , Motilidad Gastrointestinal/efectos de los fármacos , Gutatión-S-Transferasa pi/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/fisiopatología , Fosfopiruvato Hidratasa/metabolismo , Proteómica/métodos , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Estómago/fisiopatología , Superóxido Dismutasa/metabolismo , Canales Aniónicos Dependientes del Voltaje/efectos de los fármacos , Canales Aniónicos Dependientes del Voltaje/metabolismoRESUMEN
The reported data indicate that oleic acid (OA) decreases cholesterol absorption. To explore the underlying mechanisms, the effects of OA on the expression of cholesterol transport-related proteins (NPC1L1, ABCG5/8, ACAT2, MTP) and the unfolded protein response (UPR) pathway were studied in CaCo-2 enterocytes by incubating CaCo-2 cells with taurocholate micelles or taurocholate micelles containing different concentrations of OA (0.25-1.0 mM). We show that OA effectively induces XBP1 mRNA splicing, a key component of the UPR signaling, and the expression of BiP and mature ATF6 proteins in a concentration-dependent manner, leading to the induction of endoplasmic reticulum (ER) stress and activation of the UPR. Interestingly, OA decreases NPC1L1 expression in a dose-dependent manner while it has no effects on ABCG5 and MTP mRNA level or SREBP-2, ABCG8, and ACAT2 protein level. In CaCo-2 cells treated with 1.0 mM OA, both the NPC1L1 mRNA level and the NPC1L1 protein expression in brush-border membrane fractions were decreased by 39% and 37%, respectively (P < 0.01). A dose of 1 mM dithiothreitol (DTT), a positive control for ER stress induction, also decreases NPC1L1 mRNA and protein expression by 27% and 23%, respectively (P < 0.05). Furthermore, 4-phenyl-butyric acid, an UPR inhibitor, blocks OA- and DTT-induced reduction on NPC1L1 mRNA and protein levels. The results suggest that OA down-regulates NPC1L1 mRNA and protein expression via the induction of the UPR, which may play an important role in reducing intestinal cholesterol absorption.
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Colesterol/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Ácido Oléico/farmacología , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8 , Transportadoras de Casetes de Unión a ATP/metabolismo , Células CACO-2 , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Retículo Endoplásmico/efectos de los fármacos , Humanos , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero/metabolismo , Factores de Transcripción del Factor Regulador X , Esterol O-Aciltransferasa/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Factores de Transcripción/metabolismo , Proteína 1 de Unión a la X-Box , Esterol O-Aciltransferasa 2RESUMEN
This study examined the effect of insulin on the expression of very low density lipoprotein receptor (VLDLR) subtypes of SGC7901 cells and discussed its biological implication. In vitro, moderately or poorly-differentiated human gastric adenocarcinoma cell line SGC7901 was incubated with insulin for different lengths of time, and then the expression of protein and RNA level in VLDLR subtypes were detected by Western blotting and real-time PCR, respectively. The results showed that, at certain time interval, insulin could down-regulate expression of type I VLDLR and up-regulate the expression of type II VLDLR in SGC7901 cells, at both protein and RNA level. We are led to conclude that insulin serves as a regulator in maintaining the balance between glucose and lipid metabolism in vivo, possibly through its effect on the differential expression of VLDLR subtypes.
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Insulina/farmacología , Receptores de LDL/clasificación , Receptores de LDL/metabolismo , Neoplasias Gástricas/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Línea Celular Tumoral , Glucosa/metabolismo , Humanos , Metabolismo de los Lípidos , Lipoproteínas VLDL/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de LDL/genética , Neoplasias Gástricas/patologíaRESUMEN
Very low density lipoprotein receptors (VLDLR) including type I and type II are known to affect cell functions by binding to its extracellular ligands. However, the effect of these ligands on VLDLR expression remains elusive. Tissue factor pathway inhibitor (TFPI) and urokinase plasminogen activator and plasminogen activator inhibitor 1 (uPA-PAI-1) complex, two ligands of VLDLR, were used to examine their effects on VLDLR expression. TFPI treatment decreased type II VLDLR expression, inhibited cell proliferation and migration, and degradated beta-catenin in SGC7901 cells. However, uPA-PAI-1 complex, increased type II VLDLR expression with promoted cell proliferation and migration and stabilization of beta-catenin. These results indicated that extracellular ligands can change the expression of type II VLDLR to affect cell proliferation and migration.
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Lipoproteínas/farmacología , Inhibidor 1 de Activador Plasminogénico/farmacología , Receptores de LDL/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/farmacología , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos BALB C , Fosforilación/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de LDL/genética , beta Catenina/metabolismoRESUMEN
This study examined the effect of insulin on the expression of very low density lipoprotein receptor (VLDLR) subtypes of SGC7901 cells and discussed its biological implication. In vitro,moderately or poorly-differentiated human gastric adenocarcinoma cell line SGC7901 was incubated with insulin for different lengths of time, and then the expression of protein and RNA level in VLDLR subtypes were detected by Western blotting and real-time PCR, respectively. The results showed that, at certain time interval, insulin could down-regulate expression of type Ⅰ VLDLR and up-regulate the expression of type Ⅱ VLDLR in SGC7901 cells, at both protein and RNA level.We are led to conclude that insulin serves as a regulator in maintaining the balance between glucose and lipid metabolism in vivo, possibly through its effect on the differential expression of VLDLR subtypes.
RESUMEN
Reported data indicate that cholesterol loading in the liver can cause hepatic injury. To explore the possible mechanisms of cell damage resulting from cholesterol overloading in hepatocytes, cell apoptosis, the unfolded protein response (UPR) and the correlation between them were assessed in the cholesterol-overloaded normal human hepatic cell line L02. L02 cells were incubated with 200 microg/ ml of low density lipoprotein (LDL) for 24 h with or without 20 microg/ml 58035, an inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT). In the LDL+58035 group, the intracellular cholesterol level was dramatically increased, which was measured by an enzymatic combined high performance liquid chromatography assay. Expression of immunoglobulin-binding protein, X-box binding protein 1, activating transcription factor 6, activating transcription factor 4, CCAAT/enhancer-binding protein homologous protein-10, markers of endoplasmic reticulum stress (ERS)/ UPR, were up-regulated as determined using reverse transcription-polymerase chain reaction (RT-PCR) or Western blot analysis. The rate of cell apoptic death increased 21.3+/-2.4%. Meanwhile, the active caspase-3 protein expression was increased 8.4-fold compared to the active caspase-3 protein expression in the controls. Furthermore, 4-phenylbutyric acid, an inhibitor of UPR, partly reduced cell apoptosis and activation of caspase-3. This study suggests that cholesterol overloading in hepatic L02 cells induces ERS and activates the UPR which, in part, leads to the apoptotic damage of cells.
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Apoptosis/efectos de los fármacos , Colesterol/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Respuesta de Proteína Desplegada/fisiología , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 6/genética , Factor de Transcripción Activador 6/metabolismo , Western Blotting , Caspasa 3/metabolismo , Línea Celular , Proteínas de Unión al ADN/genética , Inhibidores Enzimáticos/farmacología , Humanos , Lipoproteínas LDL/farmacología , Fenilbutiratos/farmacología , Factores de Transcripción del Factor Regulador X , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esterol O-Aciltransferasa/antagonistas & inhibidores , Factores de Transcripción/genética , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
In hypertriglyceridaemic individuals, atherosclerogenesis is associated with the increased concentrations of very low density lipoprotein (VLDL) and VLDL-associated remnant particles. In vitro studies have suggested that VLDL induces foam cells formation. To reveal the changes of the proteins expression in the process of foam cells formation induced by VLDL, we performed a proteomic analysis of the foam cells based on the stimulation of differentiated THP-1 cells with VLDL. Using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis, 14 differentially expressed proteins, containing 8 up-regulated proteins and 6 down-regulated proteins were identified. The proteins are involved in energy metabolism, oxidative stress, cell growth, differentiation and apoptosis, such as adipose differentiation-related protein (ADRP), enolase, S100A11, heat shock protein 27 and so on. In addition, the expression of some selected proteins was confirmed by Western blot and RT-PCR analysis. The results suggest that VLDL not only induces lipid accumulation, but also brings about foam cells diverse characteristics by altering the expression of various proteins.
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Células Espumosas/química , Lipoproteínas VLDL/farmacología , Triglicéridos/metabolismo , Línea Celular , Electroforesis en Gel Bidimensional , Células Espumosas/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Lipoproteínas VLDL/análisis , Proteómica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Apoptin protein harbors tumor-selective cell death activity, which makes it a potential anticancer therapy candidate. This study reports an apoptin therapy approach based on protein transduction domain 4 (PTD4)-mediated transduction of recombinant apoptin protein. In vitro, the PTD4-apoptin fusion protein is located in the nucleus and induces cell death in, e.g., human hepatocarcinoma HepG2 cells. In normal human L-02 hepatocytes, PTD4-apoptin protein retained mainly cytoplasmic and did not induce detectable levels of cell death, illustrating that the PTD4 domain does not affect apoptin's tumor-selective characteristics. In vivo, liver, cervix and gastric carcinoma xenografts treated with PTD4-apoptin protein for 6 days via the tumor epidermis exhibited a significant tumor growth inhibition because of apoptin-mediated cell death. In addition, treatment of human hepatocarcinoma xenografts during 3 weeks showed that PTD4-apoptin protein has significant anticancer activity, whereas control treatment with PTD4-enhanced green fluorescence protein or saline did not. Cell death and disruption of the tumor integrity were apparent in the PTD4-apoptin transduced xenografted tumors. As important, although PTD4-apoptin protein could be detected in the epidermal tissue covering the subcutaneous tumor tissue and in several organs, such as liver and brain, of the treated mice, no tissue disruption or signs of cell death could be detected. Our in vivo data reveal that apoptin protein delivery constitutes a novel powerful and safe anticancer therapy.
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Proteínas Reguladoras de la Apoptosis/genética , Proteínas de la Cápside/metabolismo , Carcinoma Hepatocelular/terapia , Proteínas Portadoras/metabolismo , Neoplasias Hepáticas/terapia , Proteínas Recombinantes de Fusión/uso terapéutico , Animales , Proteínas de la Cápside/genética , Carcinoma Hepatocelular/patología , Proteínas Portadoras/genética , Núcleo Celular/metabolismo , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Femenino , Citometría de Flujo , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Etiquetado Corte-Fin in Situ , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Señales de Localización Nuclear , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Neoplasias Gástricas/terapia , Fracciones Subcelulares , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/terapiaRESUMEN
Very low density lipoprotein receptor (VLDLR) is thought to participate in the pathogenesis of atherosclerosis induced by VLDL and beta-VLDL. The present study was undertaken to elucidate the effects of VLDL and beta-VLDL on VLDLR expression and its signaling pathway. RAW264.7 cells were incubated with VLDL and beta-VLDL. The expression of VLDLR mRNA was detected by RT-PCR. The transcriptional activity of VLDLR gene was detected in recombinant plasmid pGL4.2VR-luciferase transfected RAW264.7. Western blot assay was used to detect the changes of phosphorylated ERK1/2 protein. Inhibitors or activators were used to observe the signal pathway involving VLDLR expression regulation. The results showed that VLDL and beta-VLDL stimulated ERK1/2 activity in a PKC-dependent manner. VLDL or beta-VLDL-induced VLDLR expression on macrophages was extremely abolished by inhibitors ERK1/2 or PKC. Our findings revealed that VLDL or beta-VLDL-induced VLDLR expression via PKC/ERK cascades and the effect was linked to the transcriptional activation of VLDLR gene promoter.
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VLDL-Colesterol/farmacología , Lipoproteínas IDL/farmacología , Macrófagos/metabolismo , Receptores de LDL/metabolismo , Línea Celular , Humanos , Macrófagos/citología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Regiones Promotoras Genéticas , Proteína Quinasa C/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de LDL/genética , Transducción de Señal , Transfección , Regulación hacia ArribaRESUMEN
AIMS: Clear differences of biological function between very low density lipoprotein receptor (VLDLR) types I and II have not been defined. The purpose of this study is to characterize VLDLR activities during cell proliferation and migration using adenocarcinoma SGC7901 cells. MAIN METHODS: Western blotting was used to test protein expression. Cell proliferation or migration was analyzed by MTT or Transwell chambers respectively. The mRNA expression was tested by RT-PCR. Reporter assay was to test the transcription activity. KEY FINDINGS: The cells treated with all-trans retinoic acid (ATRA) became well differentiated and had a gradually attenuated cell proliferation and migration, accompanied by a significant decrease in type II VLDLR expression. These cells also had decreased amount of beta-catenin, indicating a decreased stability of beta-catenin. In addition, the mRNA expression of both matrix metalloproteinase (MMP)-2 and MMP-9 was also decreased. On the contrary, cells treated with phorbol-12-myristate-13-acetate (PMA) had an increase in type II VLDLR expression, whereas the beta-catenin was increased. This was accompanied by increased cell proliferation and migration and by increased MMP-2 and MMP-9 mRNA expression. Finally, the transfection of SGC7901 cells with type II VLDLR recombinant DNA induced the cell proliferation and migration as well as the activation of beta-catenin/T cell factor (TCF) signaling. However, type I VLDLR transfection reduced beta-catenin stability and decreased cell proliferation and migration. SIGNIFICANCE: These data strongly suggest that type II VLDLR activity is positively associated with significant change of tumor cell proliferation and migration, via the beta-catenin/TCF signaling.
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Movimiento Celular/fisiología , Proliferación Celular , Receptores de LDL/metabolismo , Western Blotting , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Ligandos , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , ARN Mensajero/biosíntesis , Receptores de LDL/biosíntesis , Receptores de LDL/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Acetato de Tetradecanoilforbol/farmacología , Tretinoina/farmacología , beta Catenina/metabolismoRESUMEN
Very low density lipoprotein receptor (VLDLR) is thought to participate in the patho-genesis of atherosclerosis induced by VLDL and β-VLDL. The present study was undertaken to elu-cidate the effects of VLDL and β-VLDL on VLDLR expression and its signaling pathway. RAW264.7 cells were incubated with VLDL and β-VLDL. The expression of VLDLR mRNA was detected by RT-PCR. The transcriptional activity of VLDLR gene was detected in recombinant plasmid pGL4.2VR-luciferase transfected RAW264.7. Western blot assay was used to detect the changes of phosphorylated ERK1/2 protein. Inhibitors or activators were used to observe the signal pathway in-volving VLDLR expression regulation. The results showed that VLDL and β-VLDL stimulated ERKI/2 activity in a PKC-dependent manner. VLDL or β-VLDL-induced VLDLR expression on macrophages was extremely abolished by inhibitors ERKI/2 or PKC. Our findings revealed that VLDL or β-VLDL-induced VLDLR expression via PKC/ERK cascades and the effect was linked to the transcriptional activation of VLDLR gene promoter.
RESUMEN
In order to obtain three isoforms of apolipoprotein E (apoE), the cDNA encoding apoE3 was obtained by RT-PCR from normal human liver tissue. Site-directed mutagenesis was used to obtain the cDNAs encoding apoE2 and apoE4 isoforms. The 3 cDNAs were subcloned into vector pGEM-3Z and verified by DNA sequencing. The expression recombinant which can express the target protein as a (His) 6-tagged fusion was constructed by subcloning apoE cDNA into vector pT7-PL. The purified proteins were gained by Ni-NTA column. The SDS-PAGE results revealed the 6 His fusion proteins (apoE2, apoE3 and apoE4) were correctly expressed and purified successfully.
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Apolipoproteínas E/biosíntesis , Hígado/química , Mutación Puntual , Apolipoproteína E2 , Apolipoproteína E3 , Apolipoproteína E4 , Apolipoproteínas E/genética , Apolipoproteínas E/aislamiento & purificación , Secuencia de Bases , Clonación Molecular , Humanos , Datos de Secuencia MolecularRESUMEN
In order to construct a single chain fragment variable (ScFv) phage display library against ovarian tumor, by using RT-PCR, the human heavy chain variable region genes (VH) and light chain variable region genes (VL) were amplified from lymphocytes of ovarian tumor patients and subsequently assembled into ScFv genes by SOE. The resulting ScFv genes were electrotransformed into E. coli TG1 and amplified with the co-infection of helper phage M13KO7 to obtain phage display library. The capacity and titer of the resulting library were detected. The phage antibody library with a capacity of approximately 3 x 10(9) cfu/microg was obtained. After amplification with helper phage, the titer of antibody library reached 5 x 10(12) cfu/mL. Human ScFv library against ovarian tumor was constructed successfully, which laid a foundation for the screening of ovarian tumor specific ScFv for the radioimmunoimaging diagnosis of ovarian tumor.
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Técnicas de Visualización de Superficie Celular , Neoplasias Ováricas/genética , Anticuerpos de Cadena Única/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Neoplasias Ováricas/inmunología , Reacción en Cadena de la Polimerasa/métodos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Recombinación Genética/genética , Anticuerpos de Cadena Única/biosíntesisRESUMEN
To explore the functions of very low density lipoprotein receptor (VLDL-R) subtype II in lipoprotein metabolism and foam cells formation, the recombinant plasmid with the two subtypes cDNA was constructed respectively, the 1dl-A7 cell lines were transfected and two cell lines expressing VLDL-R were obtained: one stably expressing the VLDLR with the O-linked sugar region (type I VLDLR) and the other without the O-linked sugar region (type II VLDLR). In the study on binding of VLDLR to their nuclein labeled natural ligands (VLDL and beta-VLDL), it was found that surface binding of 25 I-VLDL or 125 I-beta-VLDL of 1dl-A7 cells transfected with type I VLDL R recombinant (1dl-A7-VRI) was more higher than that of 1dl-A7 cells transfected with type II VLDLR recombinant (1dl-A7-VRII). After being incubated with VLDL for different time, the contents of triglyceride and total cholesterol in cells were mensurated, and the formation of foam cells and accumulation of lipid in cells was observed by oil-red O staining. The results showed that the contents of triglyceride and total cholesterol in 1dl-A7-VR I were much higher than those in 1dl-A7-VR II, and 1dl-A7-VR I could transform into foam cells notably. It was suggested that type I VLDLR binds with relative higher affinity to VLDL and beta-VLDL, and internalizes much more lipoprotein into cells. As a result, we can conclude that type I VLDLR plays a more important role in lipoprotein metabolism and foam cells formation than type II VLDLR.
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Células Espumosas/citología , Lipoproteínas VLDL/metabolismo , Receptores de LDL/fisiología , Animales , Arteriosclerosis/metabolismo , Arteriosclerosis/patología , Células CHO , Células Cultivadas , Clonación Molecular , Cricetinae , Cricetulus , Células Espumosas/metabolismo , Humanos , Ligandos , Receptores de LDL/clasificación , Receptores de LDL/metabolismo , Transfección , Triglicéridos/metabolismoRESUMEN
In the present study, we examined the regulation of the expression and function of ABCA1 by modified LDL (ox-LDL) in vitro. After incubation with apoA-I for 24 h, RAW264.7 cells effluxed 37.65% cholesterol loaded by acetyl LDL (ac-LDL), and 9.78% cholesterol in ox-LDL group. The level of ABCA1 mRNA increased about three times either when cells were incubated with -100 microg /mL ac-LDL or with 100 microg /mL ox-LDL. However, the level of ABCA1 protein rose by 1.57 times in ac-LDL group and 1.26 times in ox-LDL group. These results demonstrated that ox-LDL had different effect on the expression and function of ABCA1, ox-LDL might decrease the cholesterol efflux mediated by ABCA1 through other unknown mechanisms.
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Transportadoras de Casetes de Unión a ATP/biosíntesis , Lipoproteínas LDL/farmacología , Macrófagos/efectos de los fármacos , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/fisiología , Animales , Bovinos , Línea Celular , Proliferación Celular/efectos de los fármacos , Colesterol/metabolismo , Macrófagos/metabolismo , Ratones , ARN Mensajero/biosíntesisRESUMEN
To explore the intracellular signal pathways for beta-VLDL induced very low density lipoprotein receptor (VLDL-R) transcription up-regulation and their effects on lipid accumulation in macrophages, Western Blot was used to examine phosphorylated ERK1/2 protein and regulated effects by different singal kinase inhibitants. It was found that beta-VLDL induced an increase in ERK1/2 activity in a protein kinase C (PKC)-dependent manner in murine RAW264.7 macrophages. By using different protein kinases inhibitors or activators, it was observed that the effect of beta-VLDL induced VLDL receptor transcription, which was monitored by RT-PCR analysis of VLDL receptor mRNA, was not affected by the inhibitor of p38 kinase and cAMP analog, but extremely abolished by pretreating cells with PD98059, an inhibitor of ERK and GF 109203X, an inhibitor of PKC. These results demonstrated that the PKC-ERK1/2 cascade is the essential signaling pathway by which beta-VLDL activated VLDL-R mRNA expression. Inhibition of the ERK1/2 signaling cascade resulted in suppression of the cellular lipid accumulation induced by beta-VLDL in macrophages.
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Lipoproteínas VLDL/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos/fisiología , Receptores de LDL/biosíntesis , Células Cultivadas , Macrófagos/citología , Macrófagos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Receptores de LDL/genética , Transducción de Señal , Factores de Transcripción/metabolismo , Transcripción Genética , Regulación hacia ArribaRESUMEN
The role of very low density lipoprotein receptor (LVLDR) in the process of foam cell formation was investigated. After the primary cultured mouse peritoneal macrophages were incubated with VLDL, beta-VLDL or low density lipoprotein (LDL), respectively for 24 h and 48 h, foam cells formation was identified by oil red O staining and cellular contents of triglyceride (TG) and total cholesterol (TC) were determined. The mRNA levels of LDLR, LDLR related protein (ILRP) and VLDLR were detected by semi-quantitative RT-PCR. The results demonstrated that VLDL, beta-VLDL and LDL could increase the contents of TG and TC in macrophages. Cells treated with VLDL or beta-VLDL showed markedly increased expression of VLDLR and decreased expression of LDLR, whereas LRP was up-regulated slightly. For identifying the effect of VLDL receptor on cellular lipid accumulation, ldl-A7-VR cells, which expresses VLDLR and trace amount of LRP without functional LDLR, was used to incubate with lipoproteins for further examination. The results elucidated that the uptake of triglyceride-rich lipoprotein mediated by VLDLR plays an important role in accumulation of lipid and the formation of foam cells.
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Células Espumosas/citología , Lipoproteínas VLDL/farmacología , Macrófagos Peritoneales/citología , Receptores de LDL/metabolismo , Animales , Arteriosclerosis/metabolismo , Arteriosclerosis/patología , Células Cultivadas , LDL-Colesterol/metabolismo , LDL-Colesterol/farmacología , Femenino , Células Espumosas/metabolismo , Macrófagos Peritoneales/metabolismo , Ratones , Triglicéridos/metabolismoRESUMEN
To elucidate the intracellular signaling pathways for VLDL-induced VLDLR transcription, Western blot analysis was used to examine phosphorylated ERK1/2 protein. It was found that that VLDL induced an increase in ERK1/2 activity in a protein kinase C (PKC)-dependent manner in murine RAW264.7 macrophages. By using different protein kinases inhibitors or activators it was observed that the effect of VLDL-induced VLDL receptor transcription, which is monitored by RTPCR analysis of VLDL receptor mRNA, was not affected by the inhibitor of p38 kinase and cAMP analog, but completely abolished by pretreatment of the cells with PD 98059, an inhibitor of MEK and GF 109203X, an inhibitor of PKC. These results demonstrated that the PKC/ERK1/2 cascade is the essential signaling pathway by which VLDL activates VLDL receptor mRNA expression.
Asunto(s)
Lipoproteínas VLDL/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Receptores de LDL/metabolismo , Animales , Factor de Unión a CCAAT/metabolismo , Bovinos , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Liso Vascular/metabolismo , Fosforilación , ARN Mensajero/metabolismo , Ratas , Transducción de Señal , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
In order to testify the antitumor effect, especially its effect against liver carcinoma in vivo, of VP3 protein, one kind of protein coded by chicken anemia virus, recombinants pcDNA-vp3 containing chicken anemia virus vp3 gene, and control vector pcDNA3 were mixed with murine liver carcinoma cell lines H22 respectively. The mixture was injected subcutaneously into Balb/C mice. Some days later, the mice were killed and the solid tumor weighed. The antitumor efficiency was evaluated. The manners of VP3 protein in vivo inducing tumor cell death were identified by using TUNEL assay. All the results suggested that the injection of pcDNA-vp3 and H22 mixture resulted in a significant reduction of tumor growth in mice when compared with the results of control groups. TUNEL assay revealed that VP3 induced apoptosis in vivo. All these indicated that CAV vp3 might be a potential new gene in reducing the growth rate of tumor cells in liver carcinoma or in other kind of solid tumors in vivo.