RESUMO
Lung adenocarcinoma (LUAD) is the most challenging neoplasm to treat in clinical practice. Ankyrin repeat domain 49 protein (ANKRD49) is highly expressed in several carcinomas; however, its pattern of expression and role in LUAD are not known. Tissue microarrays, immunohistochemistry, χ2 test, Spearman correlation analysis, Kaplan-Meier, log-rank test, and Cox's proportional hazard model were used to analyse the clinical cases. The effect of ANKRD49 on the LUAD was investigated using CCK-8, clonal formation, would healing, transwell assays, and nude mice experiment. Expressions of ANKRD49 and its associated downstream protein molecules were verified by real-time PCR, Western blot, immunohistochemistry, and/or immunofluorescence analyses. ANKRD49 expression was highly elevated in LUAD. The survival rate and Cox's modelling analysis indicated that there may be an independent prognostic indicator for LUAD patients. We also found that ANKRD49 promoted the invasion and migration in both in in vitro and in vivo assays, through upregulating matrix metalloproteinase (MMP)-2 and MMP-9 activities via the P38/ATF-2 signalling pathway Our findings suggest that ANKRD49 is a latent biomarker for evaluating LUAD prognosis and promotes the metastasis of A549 cells via upregulation of MMP-2 and MMP-9 in a P38/ATF-2 pathway-dependent manner.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Proteínas Musculares/metabolismo , Fator 2 Ativador da Transcrição/metabolismo , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/patologia , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Nus , Transdução de SinaisRESUMO
Lung squamous cell carcinoma (LUSC) is associated with poor clinical outcomes and identifying novel biomarkers that are involved in the progression of LUSC is important for prognosis and targeted treatment. Herein, ankyrin repeat domain 49 (ANKRD49) protein in LUSC versus paired noncancerous lung tissues was tested and its clinical significance was evaluated through χ 2 test, log-rank test, and Cox proportional hazards model. The results showed the ANKRD49 protein in LUSC was elevated and correlated with the tumor-node-metastasis stage, lymph node metastasis, distal metastasis, and differentiation. Patients with higher ANKRD49 had lower overall survival rate and higher ANKRD49 expression in lung tissues may be used as an independent prognostic marker for LUSC patients.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/patologia , Humanos , Estimativa de Kaplan-Meier , Pulmão/patologia , Neoplasias Pulmonares/patologiaRESUMO
In this paper, we developed an amplified fluorescence biosensor for acetylcholinesterase (AChE) activity detection by taking advantage of the mercury ion-mediated Mgzyme (Mg2+-dependent DNAzyme) activity. The catalytic activity of Mgzyme can be inhibited by the formation of T-Hg2+-T base pairs between the Mgzyme and mercury ions. Therefore, the Mgzyme-Hg2+ complex has no activity on a molecular beacon (MB) substrate, which afforded a very weak fluorescence background for this biosensor. After the addition of acetylcholinesterase (AChE), the substrate acetylthiocholine could be hydrolyzed to thiocholine, which has a stronger binding power with mercury ions than T-Hg2+-T base pairs. Therefore, the Mgzyme activity was recovered. The activated Mgzyme could hybridize with the MB substrate and undergo many cleavage cycles, resulting in a significant increase of fluorescence intensity. This biosensor displayed high sensitivity with the detection limit as low as 0.01 mU mL-1. Moreover, this design did not require complex composition and sequence design; thus it is simple and convenient. This biosensor was also applied for the determination of AChE in human blood and showed satisfactory results.
Assuntos
Técnicas Biossensoriais , DNA Catalítico , Mercúrio , Acetilcolinesterase/metabolismo , Técnicas Biossensoriais/métodos , DNA Catalítico/química , Humanos , Íons , Limite de Detecção , Mercúrio/químicaRESUMO
Spermatogenesis is a complicated process that is tightly regulated by the well-coordinated expression of a series of genes in the testes. Ankyrin repeat domain-containing protein 49 (ANKRD49), an evolutionarily conserved protein highly expressed in the testes, is mainly found in spermatogonia, spermatocytes, and round spermatids. However, the exact function of ANKRD49 in spermatogenesis has remained elusive. In this study, we sought to investigate the role of ANKRD49 in apoptosis and determine the mechanism underlying this process in male germ cell-derived GC-1 cells. Nuclear staining with Hoechst 33258 and annexin V-FITC/PI, as well as analysis of caspase 3 activity, mitochondrial membrane potential, and apoptotic protein expression, showed that etoposide-induced apoptosis was attenuated by ANKRD49 overexpression but promoted by RNA interference-induced ANKRD49 knockdown. Furthermore, assessment of the levels of caspase 9, caspase 8, and proteins of the Bcl-2 family revealed ANKRD49 to be involved in an intrinsic apoptosis pathway. Examination of the subcellular distribution of the NF-κB p65 subunit after treatment with an NF-κB signaling inhibitor or p65 small interfering RNA demonstrated that ANKRD49 modulated etoposide-induced GC-1 cell apoptosis via the NF-κB pathway. Taken together, these results suggest that ANKRD49 plays an important role in reducing intrinsic apoptosis of GC-1 cells by modulating the NF-κB signaling pathway.
Assuntos
Apoptose/efeitos dos fármacos , Etoposídeo/farmacologia , Células Germinativas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transativadores/metabolismo , Fator de Transcrição RelA/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular , Células Germinativas/citologia , Humanos , MasculinoRESUMO
Earthworms are useful indicator organisms of soil health and Eisenia fetida have been extensively used as test organisms in ecotoxicological studies. In order to gain insight into the gene expression profiles associated with physiological functions of earthworms, a fulllength enriched cDNA library of the Eisenia fetida genome was successfully constructed using Switching Mechanism at 5'End of RNA Template technology. Construction of a cDNA library and analysis of Expressed Sequence Tags (ESTs) are efficient approaches for collecting genomic information and identifying genes important for a given biological process. Furthermore, analysis of the expression abundance of ESTs was performed with the aim of providing genetic and transcriptomic information on the development and regenerative process of earthworms. Phrep and Crossmatch were used to process EST data and a total of 1,140 highquality EST sequences were determined by sequencing random cDNA clones from the library. Clustering analysis of sequences revealed a total of 593 unique sequences including 225 contiguous and 368 singleton sequences. Basic Local Alignment Search Tool analysis against the Kyoto Encyclopedia of Genes and Genomes database resulted in 593 significant hits (Pvalue <1x108), of which 168 were annotated through Gene Ontology analysis. The STRING database was used to determine relationships among the 168 ESTs, identifying associated genes involved in proteinprotein interactions and gene expression regulation. Based on nucleic acid and protein sequence homology, the mutual relationships between 287 genes could be obtained, which identified a portion of the ESTs as known genes. The present study reports on the construction of a highquality cDNA library representative of adult earthworms, on a preliminary analysis of ESTs and on a putative functional analysis of ESTs. The present study is expected to enhance our understanding of the molecular basis underlying the biological development of earthworms.
Assuntos
Etiquetas de Sequências Expressas , Biblioteca Gênica , Oligoquetos/genética , Análise de Sequência de DNA , Animais , Biologia Computacional/métodos , Ontologia Genética , Anotação de Sequência MolecularRESUMO
In this paper, by taking advantage of the fact that silver ions could mediate the Mg2+-dependent DNAzyme (Mgzyme) activity, we for the first time developed a turn-on fluorescent biosensor for amplified cysteine (Cys) detection. Because Mgzyme can interact with the silver ion and form cytosine-Ag+-cytosine (C-Ag+-C) base pairs, the conformation of its catalytic core was changed. As a result, the catalytic activity of Mgzyme was suppressed and the Mgzyme-Ag+ complex could not initiate the cleavage reaction. Therefore, the background fluorescence of the biosensor was very low. In the presence of Cys, Cys can bind tightly to the silver ion and disrupt the C-Ag+-C base pairs in the Mgzyme-Ag+ complex, leading to the restoration of Mgzyme activity. The activated Mgzyme could hybridize with the MB substrate and undergo many cleavage cycles, resulting in a significant increase of fluorescence intensity. This designed strategy provided amplified fluorescence detection of cysteine, with a detection limit of 2 nM. Moreover, the strong binding between Cys and Ag+ ensured that the biosensor had a desirable selectivity for Cys. This sensing system was also used to detect Cys in human urine samples and displayed satisfying results.
Assuntos
Técnicas Biossensoriais/métodos , Cisteína/urina , DNA Catalítico/metabolismo , Fluorescência , Magnésio/química , Prata/química , DNA Catalítico/química , Humanos , Limite de DetecçãoRESUMO
Club cell protein (CC16) is expressed primarily by club cells possesses antiinflammatory properties and is located in the bronchiolar epithelium. Previous studies have demonstrated that CC16 deficiency is associated with the progression of chronic obstructive pulmonary disease (COPD). In the present study, the therapeutic effects of recombinant rat CC16 protein in mice with COPD were examined and the underlying mechanisms investigated. A total of 30 adult male C57/BL6 mice were randomly divided into three groups (10 mice/group). A mouse COPD model was generated by exposing 20 mice to cigarette smoke (CS) for 24 weeks. A total of 10 mice were treated intranasally with rCC16 (2.5 µg/g body weight) and control mice were exposed to normal room air. Results indicated that rCC16 treatment ameliorated pathological damage in the lungs and reduced the production of tumor necrosis factor (TNF)α, interleukin (IL)6 and IL8, which were induced by CS exposure. After rCC16 administration, endogenous CC16 was upregulated and the body weight of COPD mice was increased, whereas the opposite was observed in CSexposed mice. Additionally, rCC16 treatment inhibited the DNA binding of NFκB/p65 in lung tissues and reduced nuclear translocation of NFκB/p65 in BALF and epithelial cells. Moreover, rCC16 treatment lead to a decrease in the total number of BALF cells, including macrophages, which was elevated in COPD mice. In conclusion, the present results demonstrate that rCC16 has therapeutic effects on COPD by downregulating proinflammatory factors via the NFκB pathway.
Assuntos
Fumar Cigarros/metabolismo , Pneumonia/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Mucosa Respiratória/metabolismo , Transdução de Sinais/efeitos dos fármacos , Uteroglobina/farmacologia , Animais , Fumar Cigarros/tratamento farmacológico , Fumar Cigarros/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Pneumonia/tratamento farmacológico , Pneumonia/patologia , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/patologia , Proteínas Recombinantes/farmacologia , Mucosa Respiratória/patologia , Fator de Transcrição RelA/metabolismoRESUMO
The present study aimed to investigate the effects of intestinal endotoxemia (IETM) in a rat model of aluminum neurotoxicity established by D-galactose and aluminum trichloride (AlCl3). Adult Wistar rats were administered Dgalactose and AlCl3 to create the aluminum neurotoxicity model. The learning and memory abilities of the rats were subsequently observed using a Morris water maze test and the serum levels of lipopolysaccharide (LPS), tumor necrosis factor (TNF)α, interleukin (IL)1, diamine oxidase (DAO), glutamine (Gln) and glutaminase were measured. The expression of S100ß in the serum was detected using an enzymelinked immunosorbent assay. The expression levels of the amyloid ßprotein (Aß) precursor (APP), presenilin 1 (PS1), ßsite APPcleaving enzyme (BACE), zona occludens protein (ZO)1 and Aß 140 in the brain of rats were detected via reversetranscription polymerase chain reaction, western blotting and immunohistochemistry. The levels of LPS, TNFα, IL1, DAO, Gln and S100ß in serum and the mRNA and protein expression levels of APP, PS1, BACE and Aß140 in the brain were markedly increased in the model rats compared with controls. The level of glutaminase in the serum and the expression of ZO1 in the brain were decreased in the model rats compared with controls. IETM was present in the rat model of aluminum neurotoxicity established by Dgalactose and AlCl3 and may be important in the development of this neurotoxicity.
Assuntos
Alumínio/toxicidade , Endotoxemia/patologia , Intestinos/patologia , Neurotoxinas/toxicidade , Cloreto de Alumínio , Compostos de Alumínio/toxicidade , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Cloretos/toxicidade , Modelos Animais de Doenças , Endotoxemia/genética , Galactose , Interleucina-1/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Lipopolissacarídeos/metabolismo , Masculino , Memória/efeitos dos fármacos , Muramidase/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Wistar , Fator de Necrose Tumoral alfa/metabolismo , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Previous studies demonstrated that herpes simplex virus thymidine kinase (HSVtk) could phosphorylate nontoxic gancyclovir (GCV) efficiently to produce phosphorylated products that result in cell apoptosis, to kill tumor cells. The present study aimed to construct a plasmid vector, pcDNA3.1pAFPTK, carrying the suicide gene driven by the alphafetoprotein (AFP) promoter, to investigate the cytotoxicity of HSVtk/GCV suicide gene system on hepatoma carcinoma cells. Reverse transcriptionpolymerase chain reaction and western blotting results demonstrated that the HSVtk gene was effectively expressed in HepG2 hepatoma carcinoma cells transfected with pcDNA3.1pAFPTK plasmid, whereas HSVtk gene expression was not detected in normal HL7702 liver cells. In addition, MTT assays indicated that cell viability of HepG2 cells with the plasmid pcDNA3.1pAFPTK decreased in a dosedependent manner following treatment with GCV for 48 h. Flow cytometry also revealed that the cell apoptosis rate and mitochondrial membrane potential reduction rate in the HepG2 cells treated with HSVtk/GCV suicide gene system were significantly higher than in the control group. Apoptosis rates in the control group and the pcDNA3.1pAFPTK group were (1.00±0.62%) and (38.70±6.03%), respectively. Mitochondrial membrane potential reduction rates in the control group and the pcDNA3.1-pAFP-TK group were (0.57±0.11%) and (22.84±5.79%), respectively. Caspase3 staining demonstrated that activated caspase3 increased significantly in the HepG2 cells treated with HSVtk/GCV suicide gene system, whereas in the control group activated caspase3 increase was not observed. The results of the present study, therefore, indicated that HSVtk suicide gene was obviously expressed in the HepG2 cells and that the HSVtk/GCV system was effective at killing HepG2 hepatoma carcinoma cells.
Assuntos
Efeito Espectador , Ganciclovir/metabolismo , Plasmídeos/genética , Pró-Fármacos , Simplexvirus/genética , Timidina Quinase/genética , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Ganciclovir/farmacologia , Regulação Viral da Expressão Gênica , Humanos , Neoplasias Hepáticas , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Simplexvirus/enzimologia , Timidina Quinase/metabolismo , alfa-Fetoproteínas/genéticaRESUMO
Clara cell protein (CC16) is an anti-inflammatory protein, which is expressed in the airway epithelium. It is involved in the development of airway inflammatory diseases, including chronic obstructive pulmonary disease and asthma. However, the exact molecular mechanism underlying its antiinflammatory action remains to be fully elucidated. The aim of the present study was to define the protein profiles of the antiinflammatory effect of CC16 in lipopolysaccharide (LPS)treated rat tracheal epithelial (RTE) cells using shotgun proteomics. Protein extracts were obtained from control RTE cells, RTE cells treated with LPS and RTE cells treated with LPS and recombinant CC16 (rCC16). Subsequent labelfree quantification and bioinformatics analyses identified 12 proteins that were differentially expressed in the three treatment groups as a cluster of five distinct groups according to their molecular functions. Five of the twelve proteins were revealed to be associated with the cytoskeleton: Matrix metalloproteinase9, myosin heavy chain 10, actinrelated protein3 homolog, elongation factor 1α1 (EF1α1), and acidic ribosomal phosphoprotein P0. Five of the twelve proteins were associated with cellular proliferation: DNAdependent protein kinase catalytic subunit, EF1α1, tyrosine 3monooxygenase, caspase recruitment domain (CARD) protein 12 and adenosylhomocysteinase (SAHH) 3. Three proteins were associated with gene regulation: EF1α1, SAHH 3 and acidic ribosomal phosphoprotein P0. Three proteins were associated with inflammation: Tyrosine 3monooxygenase, CARD protein 12 and statinrelated protein. ATPase (H+transporting, V1 subunit A, isoform 1) was revealed to be associated with energy metabolism, and uridine diphosphate glycosyltransferase 1 family polypeptide A8 with drug metabolism and detoxification. The identified proteins were further validated using reverse transcriptionquantitative polymerase chain reaction. These protein profiles, and their interacting protein network, may facilitate the elucidation of the molecular mechanisms underlying the antiinflammatory effects of CC16.
Assuntos
Células Epiteliais/metabolismo , Inflamação/genética , Proteínas Recombinantes/genética , Traqueia/metabolismo , Uteroglobina/genética , Animais , Cromatografia Líquida , Células Epiteliais/patologia , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Lipopolissacarídeos/toxicidade , Biossíntese de Proteínas/genética , Proteômica , Doença Pulmonar Obstrutiva Crônica , Ratos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Espectrometria de Massas em Tandem , Traqueia/patologia , Uteroglobina/administração & dosagem , Uteroglobina/metabolismoRESUMO
The ankyrin repeat domain 49 (ANKRD49) is an evolutionarily conserved protein highly expressed in testes. However, the function of ANKRD49 in spermatogenesis is unknown. In this study, we found that ANKRD49 resides primarily in nucleus of spermatogonia, spermatocytes and round spermatids. ANKRD49 overexpression augments starvation-induced autophagy in male germ GC-1 cells whereas shRNA knockdown of ANKRD49 attenuates the autophagy. Inhibition of NF-κB pathway by its inhibitors or p65 siRNA prevents the ANKRD49-dependent autophagy augmentation, demonstrating that ANKRD49 enhances autophagy via NF-κB pathway. Our findings suggest that ANKRD49 plays an important role in spermatogenesis via promotion of autophagy-dependent survival.
Assuntos
Repetição de Anquirina , Autofagia , Meios de Cultura Livres de Soro/farmacologia , Proteínas Musculares/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Autofagia/efeitos dos fármacos , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Masculino , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas Musculares/química , Proteínas Musculares/genética , Prófase/efeitos dos fármacos , Espermatogênese/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Testículo/efeitos dos fármacos , Testículo/metabolismo , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacosRESUMO
Nasal vaccination is an effective therapeutic regimen for preventing certain infectious diseases. The mucosal immune response is important for resistance to Toxoplasma gondii infection. In this study, we evaluated the immune responses elicited in BALB/c mice by nasal immunisation with recombinant T. gondii receptor for activated C kinase 1 (rTgRACK1) and their protective efficacy against T. gondii RH strain during both chronic and lethal infections. Nasal vaccination with rTgRACK1 increased the level of secretory IgA in nasal, intestinal and vesical washes, and the level of IFN-γ and IL-2 in intestinal washes, indicating that rTgRACK1 vaccination promotes mucosal immune responses. The mice immunised with rTgRACK1 also displayed increased levels of rTgRACK1-specific IgA, total IgG, IgG1 and in particular IgG2a in their blood sera, increased production of IFN-γ, IL-2 and IL-4 but not IL-10 from their isolated spleen cells, and enhanced splenocyte proliferation in vitro. rTgRACK1-vaccinated mice were effectively protected against infection with T. gondii RH strain, showing over 50% reduction of tachyzoite burdens in their liver and brain tissues during a chronic infection, and also a 45% increase in their survivals during a lethal challenge. These results indicate that rTgRACK1 might represent an intriguing immunogen for developing a mucosal vaccine against toxoplasmosis.
Assuntos
Antígenos de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Receptores de Superfície Celular/imunologia , Toxoplasma/imunologia , Toxoplasmose/prevenção & controle , Administração Intranasal , Animais , Antígenos de Protozoários/genética , Proliferação de Células , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Imunidade nas Mucosas , Imunoglobulina A Secretora/análise , Imunoglobulina G/sangue , Leucócitos Mononucleares/imunologia , Camundongos Endogâmicos BALB C , Vacinas Protozoárias/administração & dosagem , Vacinas Protozoárias/genética , Receptores de Quinase C Ativada , Receptores de Superfície Celular/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Baço/imunologia , Análise de Sobrevida , Toxoplasmose/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
BACKGROUND: In the field of gene therapy, viral vectors as delivery tools have a number of disadvantages for medical application. This study aimed to explore a novel nonviral vector as a vehicle for gene therapy. METHODS: Transvector-rpE-MPP and EGFP (enhanced green fluorescent protein) were used as the gene transfer carrier and the reporter gene, respectively. Polyplexes which integrate transvector-rpE-MPP, the object gene, and EGFP were formed. The optimal charge ratio, stability, and transduction capacity of the polyplexes in mouse hepatocytes in vitro and in mouse liver in vivo were investigated. The polyplexes of transvector-rpE-MPP and pcDNA(3)-EGFP, with charge ratios of 0, 0.25, 0.5, 0.75, 1 and 1.5 were compared to determine the optimal charge ratio. RESULTS: Polyplexes with charge ratios of 1:1 were most stable; pcDNA(3)-EGFP in these complexes resisted digestion by DNase I and blood plasma. On the other hand, pcDNA(3)-EGFP alone was digested. Fluorescence analysis indicated that transvector-rpE-MPP successfully delivered the reporter gene EGFP into hepatocytes and that EGFP expression was detected in hepatocyte cultures and in liver tissue. CONCLUSION: These results have laid a foundation for further study of a novel nonviral gene delivery system.
Assuntos
DNA/metabolismo , Técnicas de Transferência de Genes , Hepatócitos/metabolismo , Animais , Células Cultivadas , Terapia Genética/métodos , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hepatócitos/citologia , Histidina/genética , Histidina/metabolismo , Fígado/citologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , PlasmídeosRESUMO
BACKGROUND: The proliferation and metastasis of cancers depend on angiogenesis. This property provides the feasibility for the treatment of cancer by inhibition of angiogenesis, and many angiogenic inhibitors have been demonstrated to effectively inhibit angiogenesis and consequently the growth of solid cancer. As for the newly identified angiogenesis inhibitor, arresten, some studies have found its high activity on restrainting tumor vessel. This study was to assess the anti-angiogenic activity of arresten. METHODS: The arresten gene was obtained from a healthy puerperal's placenta tissue by the reverse transcriptase-polymerase chain reaction (RT-PCR) method, and molecular cloning to prokaryotic expression plasmid pBV220 by recombination strategy. The prokaryotic expression plasmid pBV220/arr was identified by restriction enzyme digestion and sequenced. The pBV220/arr was transformed into E. coli JM109, DH5alpha, BL21 and BL21 (DE3) by the CaCl2 transformation method. The arresten expression level was detected by SDS-PAGE. The expressed product was purified, re-naturalized and detected for its biological activity of inhibiting the angiogenesis of chorioallantoic membrane (CAM). RESULTS: The arresten gene was cloned and pBV220/arr was constructed. The arresten expression level of protein was highly increased after pBV220/arr was transformed into E. coli BL21 (DE3). SDS-PAGE showed that the expressed arresten proteins were mainly inclusion bodies and had a molecular weight of 26 kDa. The expressed arresten protein showed evident biological activities. CONCLUSIONS: The successful construction of recombinant plasmid pBV220/arr and the effective expression in E.coli have laid a foundation for further study of its anti-angiogenic function and may pave the way for future anti-tumor application.
Assuntos
Arrestina/genética , Escherichia coli/metabolismo , Expressão Gênica , Placenta/metabolismo , Plasmídeos/genética , RNA/genética , Arrestina/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Feminino , Humanos , Técnicas In Vitro , Gravidez , Células Procarióticas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
BACKGROUND: The development of a harmless and efficient nonviral gene delivery system that can facilitate the penetration of nucleic acids through the plasma membrane is a key to successful gene therapy. The aim of this study was to test a nonviral gene transferring vector's function of delivering DNA into liver cells to provide an important clue for gene transfer in liver gene therapy. METHODS: The complex of DNA and DNA delivering protein was injected into mice through their tail veins. Then the mice were killed and their liver tissue was sectioned. The gene transferring results were detected using a confocal laser scanning microscope. RESULTS: Fluorescence analysis indicated that both DNA-membrane penetrating peptide (MPP) complex and DNA- hepatocyte specific receptor binding domain (HSRBD)-MPP complex could go into liver cells. The fluorescence value of liver cells in the DNA- HSRBD-MPP group was higher than that in the DNA-MPP group. CONCLUSIONS: MPP can successfully deliver DNA and protein into cells, and MPP with a HSRBD can specifically deliver DNA into liver cells. These have laid a foundation for further study on the nonviral liver cell gene delivering system.