RESUMO
BACKGROUND: Inflammatory bowel disease (IBD) is a chronic disease of the intestinal tract in which excessive activation of inflammatory response is correlated. Cyanidin-3-O-glucoside (C3G) is a powerful anti-inflammatory agent, widely existing in fruits and vegetables. However, the role of C3G has rarely been investigated in dextran sulfate sodium (DSS)-induced colitis. METHODS: In an attempt to elucidate the possible mechanism of IBD and develop new efficient therapeutic methods for colitis, we evaluated the effects of C3G on DSS-induced colitis. DSS-induced colitic C57BL/6 mice were intraperitoneal injected with 1ug C3G or phosphate buffer every 2 days, a total of 3 times; the changes in macrophages and regular T cells were analyzed by flow cytometry and immunofluorescence. Cytokines and chemokines were measured by real-time quantitative polymerase chain reaction. RESULTS: The results showed that C3G treatment did not cause changes in body weight and colon length as much as those of DSS-treated mice only. Cytokine expression levels such as interleukin (IL)- 6, IL-1ß, IL-18, tumor necrosis factor α, interferon γ (IFN γ) in colons and mesenteric lymph nodes (mLNs) from C3G-treated mice were lower than those from colitic mice. Meanwhile, C3G injection inhibited the decrease in CCL22 levels and Tregs induction in colitic mice. Furthermore, the activation of macrophages by LPS and increase of CD169+ cells induced by type I IFN could be inhibited by C3G directly in vitro. CONCLUSIONS: The study is the first to demonstrate strong effects of C3G to alleviate DSS-induced colonic damage in mice. The effect of C3G on DSS-induced colitis clearly showed a decrease of CD169+ macrophages in both the colon and mLNs. An increase of CD169+ cells induced by type I IFN could be inhibited by C3G. All these data suggest that the role of C3G in colitic inflammation was mediated at least partially by CD169+ cells and the type I IFN pathway.
Assuntos
Antocianinas/farmacologia , Colite/prevenção & controle , Sulfato de Dextrana/toxicidade , Glucosídeos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Células Cultivadas , Quimiocina CCL22/genética , Quimiocina CCL22/metabolismo , Colite/induzido quimicamente , Colite/imunologia , Colite/patologia , Feminino , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Linfócitos T Reguladores/imunologiaRESUMO
Acquired aplastic anemia is an idiopathic paradigm of human bone marrow failure syndrome, which involves active destruction of hematopoietic stem cells and progenitors by cytotoxic T cells in the bone marrow. Aberrant expression of microRNAs in T cells has been shown to lead to development of certain autoimmune diseases. In the present study, we performed a microarray analysis of miRNA expression in bone marrow CD3+ T cells from patients with aplastic anemia and healthy controls. Overexpression of miR34a and underexpression of its target gene diacylglycerol kinase (DGK) ζ in bone marrow mononuclear cells were validated in 41 patients and associated with the severity of aplastic anemia. Further, the level of miR34a was higher in naïve T cells from patients than from controls. The role of miR34a and DGKζ in aplastic anemia was investigated in a murine model of immune-mediated bone marrow failure using miR34a-/- mice. After T-cell receptor stimulation in vitro, lymph node T cells from miR34a-/- mice demonstrated reduced activation and proliferation accompanied with a less profound down-regulation of DGKζ expression and decreased ERK phosphorylation compared to those from wild-type C57BL6 control mice. Infusion of 5 × 106 miR34a-/- lymph node T cells into sublethally irradiated CB6F1 recipients led to increased Lin-Sca1+CD117+ cells and less vigorous expansion of CD8+ T cells than injection of same number of wild-type lymph node cells. Our study demonstrates that the miR34a/DGKζ dysregulation enhances T-cell activation in aplastic anemia and targeting miR34a may represent a novel molecular therapeutic approach for patients with aplastic anemia.
Assuntos
Anemia Aplástica/genética , Diacilglicerol Quinase/genética , MicroRNAs/genética , Linfócitos T/imunologia , Anemia Aplástica/imunologia , Animais , Complexo CD3/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Ativação Linfocitária , Masculino , Camundongos , Transplante de Neoplasias , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Hepatitis B virus (HBV) infection has received increasing public attention. HBV is the prototypical member of hepadnaviruses, which naturally infect only humans and great apes and induce the acute and persistent chronic infection of hepatocytes. A large body of evidence has demonstrated that dysfunction of the host anti-viral immune response is responsible for persistent HBV replication, unresolved inflammation and disease progression. Many regulatory factors are involved in immune dysfunction. Among these, T cell immunoglobulin domain and mucin domain-3 (Tim-3), one of the immune checkpoint proteins, has attracted increasing attention due to its critical role in regulating both adaptive and innate immune cells. In chronic HBV infection, Tim-3 expression is elevated in many types of immune cells, such as T helper cells, cytotoxic T lymphocytes, dendritic cells, macrophages and natural killer cells. Tim-3 over-expression is often accompanied by impaired function of the above-mentioned immunocytes, and Tim-3 inhibition can at least partially rescue impaired immune function and thus promote viral clearance. A better understanding of the regulatory role of Tim-3 in host immunity during HBV infection will shed new light on the mechanisms of HBV-related liver disease and suggest new therapeutic methods for intervention.
Assuntos
Imunidade Adaptativa , Receptor Celular 2 do Vírus da Hepatite A/imunologia , Vírus da Hepatite B/imunologia , Hepatite B Crônica/imunologia , Imunidade Inata , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/virologia , Receptor Celular 2 do Vírus da Hepatite A/genética , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Hepatite B Crônica/genética , Hepatite B Crônica/metabolismo , Hepatite B Crônica/virologia , Interações Hospedeiro-Patógeno , Humanos , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/virologia , Polimorfismo Genético , Transdução de Sinais , Regulação para CimaRESUMO
OBJECTIVE: T cell immunoglobulin- and mucin-domain-containing molecule-4 (Tim-4) receives much attention as a potentially negative regulator of immune responses. However, its modulation on macrophages has not been fully elucidated so far. This study aimed to identify the role of Tim-4 in nitric oxide (NO) modulation. METHODS: Macrophages were stimulated with 100 ng/ml LPS or 100 U/ml IFN-γ. RT-PCR was performed to detect TIM-4 mRNA expression. Tim-4 blocking antibody and NF-κB inhibitory ligand were involved in the study. NO levels were assayed by Griess reaction. Phosphorylation of NF-κB, Jak2 or Stat1 was verified by western blot. RESULTS: Tim-4 was up-regulated in murine macrophages after interferon-gamma (IFN-γ) stimulation. Tim-4 over-expression decreased NO production and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS) or IFN-γ-stimulated macrophages. Consistently, Tim-4 blockade promoted LPS or IFN-γ-induced NO secretion and iNOS expression. Tim-4 over-expression decreased LPS-induced nuclear factor kappa B (NF-κB) p65 phosphorylation in macrophages, which was abrogated by NF-κB inhibitory ligand. On the contrary, Tim-4 blocking increased LPS-induced NF-κB signaling, which was also abrogated by NF-κB inhibition. In addition, Tim-4 blockade promoted Jak2 and Stat1 phosphorylation in IFN-γ stimulated macrophages. CONCLUSION: These results indicate that Tim-4 is involved in negative regulation of NO production in macrophages, suggesting the critical role of Tim-4 in immune related diseases.
Assuntos
Macrófagos/efeitos dos fármacos , Proteínas de Membrana/fisiologia , Óxido Nítrico/biossíntese , Animais , Linhagem Celular , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico Sintase Tipo II/metabolismo , RNA Mensageiro/genéticaRESUMO
During folliculogenesis, cumulus cells surrounding the oocyte differentiate into corona radiata cells (CRCs) and cumulus oophorus cells (COCs), which are involved in gonadal steroidogenesis and the development of germ cells. Several studies suggested that microRNAs (miRNAs) play an important regulatory role at the post-transcriptional level in cumulus cells. However, comparative miRNA profiles and associated processes in human CRCs and COCs have not been reported before. In this study, miRNA profiles were obtained from CRCs and COCs using next generation sequencing in women undergoing controlled ovarian stimulation for IVF. A total of 785 and 799 annotated miRNAs were identified in CRCs and COCs, while high expression levels of six novel miRNAs were detected both in CRCs and in COCs. In addition, different expression patterns in CRCs and COCs were detected in 72 annotated miRNAs. To confirm the miRNA profile in COCs and CRCs, quantitative real-time PCR was used to validate the expression of annotated miRNAs, differentially expressed miRNAs, and novel miRNAs. The miRNAs in the let-7 family were found to be involved in the regulation of a broad range of biological processes in both cumulus cell populations, which was accompanied by a large amount of miRNA editing. Bioinformatics analysis showed that amino acid and energy metabolism were targeted significantly by miRNAs that were differentially expressed between CRCs and COCs. Our work extends the current knowledge of the regulatory role of miRNAs and their targeted pathways in folliculogenesis, and provides novel candidates for molecular biomarkers in the research of female infertility.
Assuntos
Células do Cúmulo/metabolismo , Regulação da Expressão Gênica , MicroRNAs/genética , Oócitos/metabolismo , Adulto , Diferenciação Celular , Células do Cúmulo/citologia , Células do Cúmulo/efeitos dos fármacos , Feminino , Fertilização in vitro , Perfilação da Expressão Gênica , Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Liberador de Gonadotropina/farmacologia , Humanos , Redes e Vias Metabólicas/genética , MicroRNAs/metabolismo , Oócitos/citologia , Indução da OvulaçãoRESUMO
AIM: E-cadherin is unusually highly expressed in most ovarian cancers. This study was designed to investigate the roles of E-cadherin in the carcinogenesis and progression of ovarian cancers. METHODS: Human ovarian adenocarcinoma cell line SKOV-3 was examined. E-cadherin gene CDH1 in SKOV-3 cells was knocked down via RNA interference (RNAi), and the resultant variation of biological behavior was observed using CCK-8 and colony formation experiment. E-cadherin-mediated Ca(2+)-dependent cell-cell adhesion was used to study the mechanisms underlying the effects of E-cadherin on the proliferation and survival of SKOV-3 cells. The expression levels of E-cadherin, extracellular signal-related kinase (ERK), phosphorylated ERK (P-ERK) were measured using Western blot assays. RESULTS: Transfection with CDH1-siRNA for 24-96 h significantly suppressed the growth and proliferation of SKOV-3 cells. E-cadherin-mediated calcium-dependent cell-cell adhesion of SKOV-3 cells resulted in a rapid increase of P-ERK, but did not modify the expression of ERK protein. The phosphorylation of ERK in the cells was blocked by pretreatment with the MEK1 specific inhibitor PD98059 (50 µmol/L), but not by the PI3K inhibitor wortmannin (1 µmol/L) or PKA inhibitor H89 (10 µmol/L). CONCLUSION: E-cadherin may function as a tumor proliferation enhancer via activating the MEK/ERK pathway in development of ovarian epithelial cancers.
Assuntos
Adenocarcinoma/metabolismo , Caderinas/metabolismo , Sistema de Sinalização das MAP Quinases , Neoplasias Ovarianas/metabolismo , Adenocarcinoma/genética , Caderinas/genética , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Neoplasias Ovarianas/genética , Interferência de RNARESUMO
Programmed cell death 4 (PDCD4) is a newly identified tumor suppressor that can inhibit activator protein (AP)-1 activation and protein translation. Our previous studies indicate that lost or reduced PDCD4 expression is associated with the progression of ovarian carcinoma. However, direct evidence that PDCD4 inhibits malignant phenotype of human cancer cells is limited. In the present study, we found that PDCD4 expression in ovarian cancer cell lines (SKOV3, 3AO, and CAOV3) inhibited significantly their proliferation and cell cycle progression, and induced apoptosis. More importantly, up-regulation of PDCD4 expression decreased the colony-forming capacity of ovarian cancer cells in vitro and tumorigenic capacity in mice. These results demonstrate that PDCD4 can suppress the malignant phenotype of ovarian cancer cells, and may represent a novel therapeutic target for the treatment of ovarian cancer.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Carcinoma/patologia , Neoplasias Ovarianas/patologia , Proteínas de Ligação a RNA/metabolismo , Animais , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/fisiologia , Carcinoma/genética , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Neoplasias Ovarianas/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Distribuição Aleatória , Fator de Transcrição AP-1/antagonistas & inibidores , Transfecção , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) has been reported to participate in the pathogenesis of inflammatory diseases. However, whether Tim-3 is involved in hepatitis B virus (HBV) infection remains unknown. Here, we studied the expression and function of Tim-3 in a hydrodynamics-based mouse model of HBV infection. A significant increase of Tim-3 expression on hepatic T lymphocytes, especially on CD8+ T cells, was demonstrated in HBV model mice from day 7 to day 18. After Tim-3 knockdown by specific shRNAs, significantly increased IFN-gamma production from hepatic CD8+ T cells in HBV model mice was observed. Very interestingly, we found Tim-3 expression on CD8+ T cells was higher in HBV model mice with higher serum anti-HBs production. Moreover, Tim-3 knockdown influenced anti-HBs production in vivo. Collectively, our data suggested that Tim-3 might act as a potent regulator of antiviral T-cell responses in HBV infection.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Hepatite B/imunologia , Interferon gama/biossíntese , Fígado/imunologia , Receptores Virais/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Receptor Celular 2 do Vírus da Hepatite A , Interferon gama/imunologia , Fígado/patologia , Fígado/virologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Receptores Virais/genéticaRESUMO
Endostatin can inhibit tumor growth by blocking angiogenesis, whereas tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) may function as a soluble cytokine to selectively kill cancer cells without toxicity to most normal cells. To establish the combined anti-tumor therapeutic effect of endostatin and soluble TRAIL (sTRAIL), we performed intra-tumoral human endostatin and sTRAIL gene transfer using plasmid pVAX1 as a vector in a nude mouse model of human liver cancer. For subcutaneously inoculated human BEL7402 cancer, co-expression of both transgenes conferred marked anti-tumor activity with a significant reduction in tumor vessel density and an increase in apoptotic rates, which was accompanied with a strong activation of caspase-3. Importantly, combination therapy employing one-half dose of endostatin and sTRAIL plasmids was more effective than single endostatin or sTRAIL therapy. These results indicate that a pVAX1-mediated combinatorial antiangiogenic and proapoptotic gene therapy approach involving endostatin and sTRAIL can be an effective novel form of treatment for human liver cancer.
Assuntos
Carcinoma Hepatocelular/terapia , Endostatinas/metabolismo , Neoplasias Hepáticas Experimentais/terapia , Neovascularização Patológica/terapia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Apoptose , Western Blotting , Células COS , Carcinoma Hepatocelular/irrigação sanguínea , Carcinoma Hepatocelular/patologia , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Endostatinas/genética , Citometria de Fluxo , Terapia Genética/métodos , Humanos , Marcação In Situ das Extremidades Cortadas , Neoplasias Hepáticas Experimentais/irrigação sanguínea , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Plasmídeos/genética , Ligante Indutor de Apoptose Relacionado a TNF/genética , Transfecção , Resultado do Tratamento , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: To study the interrelation between the change of temperature and Ca2+-, Mg2+ -ATPase activity in the tissues along the running course of meridians in the rabbit. METHODS: Seven healthy mongrel rabbits were used in this study. Before and after moxibustion of "Zusanli" (ST36) and "Yinlingquan" (SP9), the cutaneous temperature along the Stomach Meridian and Spleen Meridian was detected with an infra-red thermography. The tissues showing higher temperature (including skin, subcutaneous tissue and muscle, 100 mg) and non-higher temperature (skin, subcutaneous tissue and muscle about 0.5 cm beside the higher temperature region, 100 mg) after moxibustion were sampled respectively for detecting the activity of Ca2+- and Mg2+ -ATPase with enzymologic method. RESULTS: After moxibustion of "Zusanli" (ST36) and 'Yinlingquan" (SP9), the temperature of the skin along the Stomach Meridian and Spleen Meridian on the same side increased significantly (P<0.05, 0.01); correspondingly, the activity of Mg2+ -ATPase in the higher temperature regions (lateral side of the left thigh, and the medial side of the right hindlimb) increased moderately and significantly separately in comparison with the control regions (P<0.05). No marked changes were found in the activity of Ca2+ -ATPase in the same tissues of the higher temperature regions (P>0.05). CONCLUSION: In the tissues of higher temperature region after moxibustion of SP9, Mg2+ -ATPase activity increased evidently, that may contribute to the increase of temperature along the skin of the Spleen Meridian after moxibustion, while Ca2+ -ATPase may play a minor role therein.
Assuntos
ATPase de Ca(2+) e Mg(2+)/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Meridianos , Temperatura Cutânea , Pontos de Acupuntura , Animais , Metabolismo Energético , Feminino , Masculino , CoelhosRESUMO
OBJECTIVE: To study the inhibitory effect of endostatin mediated by lipofectin on transplanted ovarian cancer in nude mice. METHODS: Constructed recombinant vector pVAX1-sEn expressing human endostatin protein was transfected into ovarian cancer cell line 3AO by lipofectin. mRNA of endostatin was detected by RT-PCR. The expression of endostatin in supernatants was detected by enzyme-linked immunosorbent assay (ELISA). The inhibitory effect of pVAX1-sEn on endothelial cell line ECV-204 was detected by methyl thiazolyl tetrazolium (MTT). By use of lipofectin mediated pVAX1-sEn for intratumor injection, the inhibitory effect on growth of ovarian cancer was observed. RESULTS: The result of RT-PCR showed there was a specific band at 610 bp. The expression quantity of endostatin in transfected cell supernatant was (201 +/- 8) ng/ml by ELISA. MTT showed pVAX1-sEn transfected cell supernatant could effectively inhibit the growth of ECV-204, the highest inhibitory ratio was 42%. The tumor volumes in pVAX1-sEn treatment group was (0.85 +/- 0.18) cm(3), significantly smaller than that in normal saline control group (1.90 +/- 0.28) cm(3) and pVAX1 control group (1.78 +/- 0.32) cm(3) (P < 0.05). HE stain in tumor tissue showed that there were obvious necrosis cells in the pVAX1-sEn treatment group, but there were flourishly growing tumor cells in pVAX1 and normal saline control groups. CONCLUSION: pVAX1-sEn mediated by lipofectin can effectively inhibit the growth of ovarian cancer.
Assuntos
Proliferação de Células , Regulação para Baixo , Endostatinas/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/fisiopatologia , Animais , Linhagem Celular Tumoral , Endostatinas/genética , Feminino , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Neoplasias Ovarianas/genética , Fosfatidiletanolaminas , Distribuição Aleatória , TransfecçãoRESUMO
AIM: To explore a safe and efficient strategy of tumor therapy using anti-angiogenetic agents. METHODS: Endostatin gene with a signal sequence of human IgGgamma chain was amplified by PCR and cloned into pVAX1 plasmid which was the only vector authorized by FDA in clinical trial to construct a recombinant plasmid named as pVAX-sEN. The recombinant plasmid was detected with EcoRI/KpnI and DNA sequencing. BALB/c mice bearing hepatocarcinoma cell line H22 were treated with naked pVAX-sEN or liposome-DNA complex in which the dose of DNA and the ratio of DNA and liposome were different from each other. To compare the efficiency of gene transfection, expression of endostatin at the treated tumor site was assayed with ELISA. To investigate the effect of pVAX1-sEN on hepatocellular carcinoma, pVAX-sEN either naked or in liposome-DNA complex was injected into BALB/c mice bearing H22, then the diameter of tumors was measured, microvessel density was detected by immunohistochemistry, endostatin expression in vivo was assayed at different time points. RESULTS: DNA sequencing showed the endostatin gene with the signal peptide was correctly cloned. In situ gene expression assay indicated that both the ratio of DNA and liposome and the dose of DNA could affect the gene transfection efficiency. Interestingly, naked pVAX-sEN had a similar in situ endostatin expression to pVAX-sEN with liposome. Animal experiments showed that pVAX-sEN together with pVAX-sEN-liposome complex could efficiently suppress the growth of mouse hepatoma cells. CONCLUSION: Naked endostatin plasmid intratumoral injection can get a similar gene transfection efficiency to liposome-DNA complex when used in situ.
Assuntos
Endostatinas/genética , Animais , Sequência de Bases , Carcinoma Hepatocelular , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Primers do DNA , Endostatinas/sangue , Endostatinas/metabolismo , Endostatinas/farmacologia , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Humanos , Lipossomos/farmacologia , Neoplasias Hepáticas , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/sangue , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
AIM: To establish a mice model of hepatitis B by using HBV-transgenic mice, and to transfer HBV-specific cytotoxic T lymphocytes (CTL) induced from syngeneic BALB/c mice immunized by a eukaryotic expression vector containing HBV complete genome DNA. METHODS: HBV DNA was obtained from digested pBR322-2HBV and ligated with the vector pcDNA3. Recombinant pcDNA3-HBV was identified by restriction endonuclease assay and transfected into human hepatoma cell line HepG2 with lipofectin. ELISA was used to detect the expression of HBsAg in culture supernatant, and RT-PCR to determine the existence of HBV PreS1 mRNA. BALB/c mice were immunized with pcDNA3-HBV or pcDNA3 by intramuscular injection. ELISA was used to detect the expression of HBsAb in serum. MTT assay was used to measure non-specific or specific proliferation ability and specific killing activity of spleen lymphocytes. Lymphocytes from immunized mice were transferred into HBV-transgenic mice (2.5X10(7) per mouse). Forty-eight hours later, the level of serum protein and transaminase was detected with biochemical method, liver and kidney were sectioned and stained by HE to observe the pathological changes. RESULTS: By enzyme digestion with Eco RI, Xho I and Hind III, the recombinant pcDNA3-HBV was verified to contain a single copy of HBV genome, which was inserted in the positive direction. HepG2 cells transfected with the recombinant could stably express PreS1 mRNA and HBsAg. After immunized by pcDNA3-HBV for 4 weeks, HBsAb was detected in the serum of BALB/c mice. The potential of spleen lymphocytes for both non-specific and specific proliferation and the specific killing activity against target cells were enhanced. The transgenic mice in model group had no significant changes in the level of serum protein but had an obvious increase of ALT and AST. The liver had obvious pathological changes, while the kidney had no evident damage. CONCLUSION: A eukaryotic expression vector pcDNA3-HBV containing HBV complete genome is constructed successfully. HepG2 cells transfected with the recombinant can express PreS1 mRNA and HBsAg stably. Specific cellular immune response can be induced in mice immunized by pcDNA3-HBV. A mice model of acute hepatitis with HBV has been established.
Assuntos
DNA Viral , Modelos Animais de Doenças , Vírus da Hepatite B/genética , Hepatite B , Camundongos Transgênicos , Linfócitos T Citotóxicos/transplante , Animais , Linhagem Celular Tumoral , Genoma Viral , Hepatite B/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T Citotóxicos/imunologia , TransfecçãoRESUMO
AIM: To evaluate the inhibitory effect of antisense phosphorothioate oligonucleotide (asON) complementary to the initiator of human telomerase catalytic subunit (hTERT) on the growth of hepatoma cells. METHODS: The as-hTERT was synthesized by using a DNA synthesizer. HepG2.2.15 cells were treated with as-hTERT at the concentration of 10 micromol/L. After 72 h, these cells were obtained for detecting growth inhibition, telomerase activity using the methods of MTT, TRAP-PCR-ELISA, respectively. BALB/c(nu/nu) mice were injected HepG2.2.15 cells and a human-nude mice model was obtained. There were three groups for anti-tumor activity study. Once tumors were established, these animals in the first group were administered as-hTERT and saline. Apoptosis of tumor cells was detected by FCM. In the 2nd group, the animals were injected HepG2.2.15 cells together with as-hTERT. In the third group, the animals were given as-hTERT 24 hours postinjection of HepG2.2.15 cells. The anti-HBV effects were assayed with ELISA in vitro and in vivo. RESULTS: Growth inhibition was observed in cells treated with as-hTERT in vitro. A significant different in the value of A570-A630 was found between cells treated with as-hTERT and control (P<0.01) by MTT method. The telomerase activity of tumor cells treated with as-hTERT was reduced, the value of A450 nm was 0.42 compared to control (1.49) with TRAP-PCR-ELISA. The peak of apoptosis in tumor cells given as-hTERT was 21.12%, but not seen in saline-treated control. A prolonged period of carcinogenesis was observed in the second and third group animals. There was inhibitory effect on the expression of HBsAg and HBeAg in vivo and in vitro. CONCLUSION: As-hTERT has an anti-tumor activity, which may be useful for gene therapy of tumors.
Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas Experimentais/patologia , Oligonucleotídeos Antissenso/farmacologia , Telomerase/genética , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Transplante de NeoplasiasRESUMO
OBJECTIVE: To study the specific expression of the antisense RNA against hepatitis B virus X (HBX) gene in hepatoblastoma cell line and its anti -HBV activity. METHODS: HBX gene (nt.1370-1827) was amplified by PCR, then cloned into EB virus vector pEBAF which contained human alpha-fetoprotein promoter and enhancer. After transfected into 2.2.15 hepatoma cells and ECV304 human endothelial cells by lipofectin, northern blot, ELISA and real-time qualitative PCR were carried out to assay the expression of HBX mRNA, HBV antigens and HBV DNA level, respectively. RESULTS: The HBX antisense RNA expression vector pEBAF-as-HBX which could be expressed specifically in 2.2.15 hepatoblastoma cells was successfully constructed. Both HBV DNA level and the expressions of hepatitis B virus surface antigen (HBsAg) and e antigen (HBeAg) in 2.2.15 hepatoblastoma cells were inhibited by pEBAF-as-HBX. Compared with those in sense control (pEBAF-s-HBX), the inhibitory rates of HBsAg, HBeAg, and HBV DNA were 37.9%, 36.8%, and 25%, respectively. CONCLUSIONS: The pEBAF-as-HBX expression vector may lead to targeted-expression of HBX antisense RNA in hepatoma cells and shows great inhibition effect on HBV.
Assuntos
Carcinoma Hepatocelular/virologia , Vírus da Hepatite B/genética , Neoplasias Hepáticas/virologia , RNA Antissenso/farmacologia , Transativadores/biossíntese , alfa-Fetoproteínas/genética , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Replicação do DNA , Elementos Facilitadores Genéticos/genética , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Terapia Genética/métodos , Vírus da Hepatite B/fisiologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Regiões Promotoras Genéticas/genética , Transativadores/genética , Ativação Transcricional , Transfecção , Proteínas Virais Reguladoras e AcessóriasRESUMO
AIM: To construct a novel HBV antisense RNA delivery system targeting hapatocellular carcinoma and study its inhibitory effect in vitro and in vivo. METHODS: GE7,a 16-peptide specific to EGFR, and HA20,a homologue of N-terminus of haemagglutinin of influenza viral envelope protein, were synthesized and conjugated with polylysin. The above conjugates were organized into the pEBAF-as-preS2, a hepatocarcinoma specific HBV antisense expression vector, to construct a novel HBV antisense RNA delivery system, named AFP-enhancing 4-element complex. Hepatocelluar carcinoma HepG2.2.15 cells was used to assay the in vitro inhibition of the complex on HBV. Expression of HBV antigen was assayed by ELISA. BALB/c nude mice bearing HepG2.2.15 cells were injected with AFP-enhancing 4-element complex. The expression of HBV antisense RNA was examined by RT-PCR and the size of tumor in nude mice were measured. RESULTS: The AFP-enhancing 4-element complex was constructed and DNA was completely trapped at the slot with no DNA migration when the ratio of polypeptide to plasmid was 1:1. The expression of HBsAg and HBeAg of HepG2.2.15 cells was greatly decreased after being transfected by AFP-enhancing 4-element complex. The inhibitory rates were 33.4 % and 58.5 % respectively. RT-PCR showed HBV antisense RNA expressed specifically in liver tumor cells of tumor-bearing nude mice. After 4 injections of AFP-enhancing 4-element complex containing 0.2 micro g DNA, the diameter of the tumor was 0.995 cm+/-0.35, which was significantly smaller than that of the control groups(2.215 cm+/-0.25, P<0.05). CONCLUSION: AFP-enhancing 4-element complex could deliver HBV antisense RNA targeting on hepatocarcinoma and inhibit both HBV and liver tumor cells in vitro and in vivo.
Assuntos
Carcinoma Hepatocelular/genética , Técnicas de Transferência de Genes , Vírus da Hepatite B/genética , Neoplasias Hepáticas/genética , RNA Antissenso , RNA Viral/genética , Animais , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Células Tumorais CultivadasRESUMO
AIM: To detect the expression of soluble TRAIL (TNF-related apoptosis inducing ligand, TRAIL) in the peripheral blood of HBV infected patients and try to elucidate whether the expression level of sTRAIL have any correlativity with the clinical staging, the expression level of HBV markers and the degree of liver damage. METHODS: 52 cases of HBV infected patients were investigated, including 8 HBV carriers, 30 chronic hepatitis B, 11 cirrhotics and 3 HBV infection related hepatocellular carcinoma. Expression of soluble TRAIL and markers of the hepatitis B were measured by enzyme-linked immunosorbent assay. RESULTS: The expression level of sTRAIL in the peripheral blood of the HBV infected patients was significantly higher than that of healthy controls (1378.35+/-540.23 pg/ml vs 613.75+/-175.80 pg/ml, P<0.001). In the group of chronic hepatitis, the expression level of sTRAIL was coincident with the status of the disease and was significantly correlated with the level of ALT. In the group of cirrhosis and liver cancer, its expression level was significantly higher than that of the healthy persons and HBV carriers, but lower than that of the hepatitis B patients; meanwhile, the expression of sTRAIL did not have any correlativity with the functional indexes of the liver. CONCLUSION: The soluble TRAIL in the HBV infected people may participate in the liver damage. Our results indicated that the expression level of soluble TRAIL may reflect the ravage of liver caused by host immune reaction to a certain degree.