RESUMO
The heterogeneous nature of tumour microenvironment (TME) underlying diverse treatment responses remains unclear in nasopharyngeal carcinoma (NPC). Here, we profile 176,447 cells from 10 NPC tumour-blood pairs, using single-cell transcriptome coupled with T cell receptor sequencing. Our analyses reveal 53 cell subtypes, including tumour-infiltrating CD8+ T, regulatory T (Treg), and dendritic cells (DCs), as well as malignant cells with different Epstein-Barr virus infection status. Trajectory analyses reveal exhausted CD8+ T and immune-suppressive TNFRSF4+ Treg cells in tumours might derive from peripheral CX3CR1+CD8+ T and naïve Treg cells, respectively. Moreover, we identify immune-regulatory and tolerogenic LAMP3+ DCs. Noteworthily, we observe intensive inter-cell interactions among LAMP3+ DCs, Treg, exhausted CD8+ T, and malignant cells, suggesting potential cross-talks to foster an immune-suppressive niche for the TME. Collectively, our study uncovers the heterogeneity and interacting molecules of the TME in NPC at single-cell resolution, which provide insights into the mechanisms underlying NPC progression and the development of precise therapies for NPC.
Assuntos
Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/metabolismo , Carcinoma Nasofaríngeo/imunologia , Carcinoma Nasofaríngeo/metabolismo , Microambiente Tumoral/fisiologia , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Microambiente Tumoral/imunologiaRESUMO
Oncolytic viruses (OV) have shown excellent safety and efficacy in preclinical and clinical studies. Influenza A virus (IAV) is considered a promising oncolytic virus. In this report, we generated a recombinant influenza virus expressing an immune checkpoint blockade agent targeting CTLA4. Using reverse genetics, a recombinant influenza virus, termed rFlu-CTLA4, encoding the heavy chain of a CTLA4 antibody on the PB1 segment and the light chain of the CTLA4 antibody on the PA segment was produced. RFlu-CTLA4 could replicate to high titers, and antibodies were produced in the allantoic fluid of infected eggs. Furthermore, the selective cytotoxicity of the virus was higher in various hepatocellular carcinoma cancer cell lines than in the normal cell line L02 in vitro, as indicated by MTS assays. More importantly, in a subcutaneous H22 mouse hepatocarcinoma model, intratumoral injections of rFlu-CTLA4 inhibited the growth of treated tumors and increased the overall survival of mice compared with injections of the PR8 virus. Taken together, these results warrant further exploration of this novel recombinant influenza virus for its potential use as a single or combination agent for cancer immunotherapy.
Assuntos
Antígeno CTLA-4/imunologia , Imunoterapia/métodos , Vírus da Influenza A/imunologia , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/imunologia , Animais , Células Hep G2 , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: Accumulating evidence has revealed that several long non-coding ribonucleic acids (lncRNAs) are crucial in the progress of hepatocellular carcinoma (HCC). AIM: To classify a long non-coding RNA, i.e., lncRNA W5, and to determine the clinical significance and potential roles of lncRNA W5 in HCC. METHODS: The results showed that lncRNA W5 expression was significantly downregulated in HCC cell lines and tissues. Analysis of the association between lncRNA W5 expression levels and clinicopathological features suggested that low lncRNA W5 expression was related to large tumor size (P < 0.01), poor histological grade (P < 0.05) and serious portal vein tumor thrombosis (P < 0.05). Furthermore, Kaplan-Meier survival analysis showed that low expression of lncRNA W5 predicts poor overall survival (P = 0.016). RESULTS: Gain-of-loss function experiments, including cell counting kit8 assays, colony formation assays, and transwell assays, were performed in vitro to investigate the biological roles of lncRNA W5. In vitro experiments showed that ectopic overexpression of lncRNA W5 suppressed HCC cell proliferation, migration and invasion; conversely, silencing of lncRNA W5 promoted cell proliferation, migration and invasion. In addition, acting as a tumor suppressor gene in HCC, lncRNA W5 inhibited the growth of HCC xenograft tumors in vivo. CONCLUSION: These results showed that lncRNA W5 is down-regulated in HCC, and it may suppress HCC progression and predict poor clinical outcomes in patients with HCC. LncRNA W5 may serve as a potential HCC prognostic biomarker in addition to a therapeutic target.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , RNA Longo não Codificante , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Prognóstico , RNA Longo não Codificante/genéticaRESUMO
BACKGROUND: Imported malaria has been an important challenge for China. Fatality rates from malaria increased in China, particularly in Henan Province, primarily due to malpractice and misdiagnoses in healthcare institutions, and the level of imported malaria. This study aims to investigate the relationship between the state of diagnosis and subsequent complications among imported malaria cases at healthcare institutions, based on malaria surveillance data in Henan Province from 2012 to 2017. METHODS: A retrospective descriptive analysis was performed using data from the Centre for Disease Control and Prevention, Zhengzhou City, the capital of Henan Province. A decision tree method was exploited to provide valuable insight into the correlation between imported malaria cases and healthcare institutions. RESULTS: From 2012 to 2017, there were 371 imported malaria cases, mostly in males aged between 20 and 50 years, including 319 Plasmodium falciparum cases. First visits of 32.3%, 19.9% and 15.9% malaria cases for treatment were to provincial, municipal and county healthcare institutions, respectively. The time interval between onset and initial diagnosis of 284 cases (76.5%) and the time interval between initial diagnosis and final diagnosis of 197 cases (53.1%) was no more than 72 h. An apparent trend was found that there were notably fewer patients misdiagnosed at first visit to healthcare institutions of a higher administrative level; 12.5% of cases were misdiagnosed in provincial healthcare institutions compared to 98.2% in private clinics, leading to fewer complications at healthcare institutions of higher administrative level due to correct initial diagnosis. In the tree model, the rank of healthcare facilities for initial diagnosis, and number of days between onset and initial diagnosis, made a major contribution to the classification of initial diagnosis, which subsequently became the most significant factor influencing complications developed in the second tree model. The classification accuracy were 82.2 and 74.1%, respectively for the tree models of initial diagnosis and complications developed. CONCLUSION: Inadequate seeking medical care by imported malaria patients, and insufficient capacity to diagnose malaria by healthcare institutions of lower administrative level were identified as major factors influencing complications of imported malaria cases in Henan Province. The lack of connection between uncommon imported malaria cases and superior medical resources was found to be the crucial challenge. A web-based system combined with WeChat to target imported malaria cases was proposed to cope with the challenge.
Assuntos
Doenças Transmissíveis Importadas/prevenção & controle , Árvores de Decisões , Instalações de Saúde , Malária/prevenção & controle , Adolescente , Adulto , Idoso , China , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Plasmodium/fisiologia , Estudos Retrospectivos , Adulto JovemRESUMO
Long non-coding RNAs (lncRNAs) have been investigated as a novel class of regulators of cellular processes, including cell growth, apoptosis and carcinogenesis. lncRNA BRAF-activated non-protein coding RNA (BANCR) has recently been revealed to be involved in tumorigenesis of numerous types of cancer, including papillary thyroid carcinoma, melanoma, non-small cell lung cancer and colorectal cancer. However, the expression profiles and biological relevance of lncRNA BANCR in hepatocellular carcinoma (HCC) has not yet been reported. In the present study, the expression level of BANCR in tumor tissues and para-cancerous tissues was determined by reverse transcription-quantitative polymerase chain reaction in patients with hepatitis B virus (HBV)-associated HCC, and its association with clinicopathological characteristics of patients was analyzed. The results demonstrated that the expression level of BANCR was significantly reduced in tumor tissues in comparison with in para-cancerous tissues (P<0.001). Furthermore, the present study demonstrated that BANCR expression level was closely associated with serum α-fetoprotein levels (P<0.01) and HCC tumor number (P<0.05). To the best of our knowledge, these results revealed for the first time that BANCR downregulated in patients with HBV-associated HCC and BANCR expression level may be a potential valuable diagnosis and therapeutic biomarker in HCC.
RESUMO
Hepatocellular carcinoma (HCC) is a leading cause of cancer-associated mortality worldwide. Despite progress in the diagnosis and treatment of HCC, prognosis remains unfavorable. Long non-coding RNAs (lncRNAs) are emerging as important factors in tumorigenesis and cancer progression; however, the underlying molecular mechanisms and clinical significance of lncRNAs in HCC remain largely unknown. The present study examined the expression pattern and clinical significance of a novel lncRNA, LOC728290, in HCC. Expression of LOC728290 was markedly decreased in HCC tissues compared with adjacent non-tumor liver tissues, as detected using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The area under the receiver operating characteristic curve for LOC728290 was 0.728. The expression of LOC728290 was associated with the level of α-fetoprotein and microvascular invasion. Furthermore, patients with low LOC728290 expression exhibited decreased recurrence-free survival times (P<0.05) compared with those with high LOC728290 expression. The results of the present study indicated that downregulation of LOC728290 in patients with HCC may be a powerful tumor biomarker, with potential clinical applications in prognosis as well as a therapeutic target.
RESUMO
The highly pathogenic avian influenza (HPAI) H5N1 virus has caused several outbreaks in domestic poultry. Despite great efforts to control the spread of this virus, it continues to evolve and poses a substantial threat to public health because of a high mortality rate. In this study, we sequenced whole genomes of eight H5N1 avian influenza viruses isolated from domestic poultry in eastern China and compared them with those of typical influenza virus strains. Phylogenetic analyses showed that all eight genomes belonged to clade 2.3.2.1 and clade 7.2, the two main circulating clades in China. Viruses that clustered in clade 2.3.2.1 shared a high degree of homology with H5N1 isolates located in eastern Asian. Isolates that clustered in clade 7.2 were found to circulate throughout China, with an east-to-west density gradient. Pathogenicity studies in mice showed that these isolates replicate in the lungs, and clade 2.3.2.1 viruses exhibit a notably higher degree of virulence compared to clade 7.2 viruses. Our results contribute to the elucidation of the biological characterization and pathogenicity of HPAI H5N1 viruses.
Assuntos
Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/virologia , Animais , China , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/patogenicidade , Camundongos Endogâmicos BALB C , Filogenia , Aves DomésticasRESUMO
BACKGROUND: Streptococcus suis is an important swine pathogen and zoonotic agent. Infection with this highly pathogenic strain can cause streptococcal toxic shock-like syndrome (STSLS), characterized by a Th-1 inflammatory cytokine storm, and a high mortality rate. Monocyte derived dendritic cells (MoDCs) are known to stimulate Th-1 cell differentiation, but the role of MoDCs in STSLS remains to be elucidated. METHODOLOGY AND FINDINGS: Porcine CD14-positive monocytes, purified from peripheral blood mononuclear cells (PBMCs), were used to generate MoDCs using granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Highly pure MoDCs were generated, as proved by their morphology, phenotype analysis, phagocytic ability, and induction of T cells proliferation. The MoDCs were further stimulated by the virulent S. suis serotype 2 (SS2) SC19 strain which triggered a strong release of several pro-inflammatory cytokines, including IL-1ß, IL-8, TNF-α, IFN-γ, and IL-12. Furthermore, the stimulated MoDCs induced CD4+ T cell differentiation towards Th-1 cells in vitro. CONCLUSIONS: The results of this study indicated that the porcine MoDCs stimulated by SS2 could release high levels of Th-1 inflammatory cytokines and induce CD4+ T cell differentiation towards Th-1 cells. Hence, it is likely that porcine MoDCs play an important role in the STSLS caused by SS2.
Assuntos
Células Dendríticas/imunologia , Monócitos/imunologia , Infecções Estreptocócicas/imunologia , Streptococcus suis/imunologia , Doenças dos Suínos/imunologia , Células Th1/imunologia , Animais , Diferenciação Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Citocinas/imunologia , Inflamação/imunologia , Inflamação/microbiologia , Infecções Estreptocócicas/veterinária , Suínos , Doenças dos Suínos/microbiologiaRESUMO
5-Hydroxymethylcytosine (5hmC), a modified form of cytosine that is considered the sixth nucleobase in DNA, has been detected in mammals and is believed to play an important role in gene regulation. In this study, 5hmC modification was detected in rice by employing a dot-blot assay, and its levels was further quantified in DNA from different rice tissues using liquid chromatography-multistage mass spectrometry (LC-MS/MS/MS). The results showed large intertissue variation in 5hmC levels. The genome-wide profiles of 5hmC modification in three different rice cultivars were also obtained using a sensitive chemical labelling followed by a next-generation sequencing method. Thousands of 5hmC peaks were identified, and a comparison of the distributions of 5hmC among different rice cultivars revealed the specificity and conservation of 5hmC modification. The identified 5hmC peaks were significantly enriched in heterochromatin regions, and mainly located in transposable elements (TEs), especially around retrotransposons. The correlation analysis of 5hmC and gene expression data revealed a close association between 5hmC and silent TEs. These findings provide a resource for plant DNA 5hmC epigenetic studies and expand our knowledge of 5hmC modification.
Assuntos
Citosina/análogos & derivados , DNA de Plantas/genética , Epigênese Genética , Oryza/genética , 5-Metilcitosina/análogos & derivados , Cromatografia Líquida , Mapeamento Cromossômico , Citosina/metabolismo , Elementos de DNA Transponíveis , DNA de Plantas/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Oryza/metabolismo , Análise de Sequência de DNA , Espectrometria de Massas em TandemRESUMO
MicroRNA (miRNA) is a kind of short non-coding RNA, involved in various cellular processes. During keratinocyte differentiation, miRNAs act as important regulators. In this study, we demonstrated by microarray assay that the expression of miR-378b significantly increased during keratinocytes differentiation. Our findings showed that miR-378b could inhibit proliferation, migration and differentiation in keratinocytes. Luciferase reporter assays showed that miR-378b directly target NKX3.1. Silencing of NKX3.1 could coincide with the effects of miR-24 overexpression. In conclusion, our results demonstrate miR-378b promote keratinocytes differentiation by targeting NKX3.1. Manipulation of miR-378b may afford a new strategy to clinic treatment of skin injury and repair.
Assuntos
Diferenciação Celular , Proteínas de Homeodomínio/metabolismo , Queratinócitos/citologia , MicroRNAs/genética , Pele/citologia , Fatores de Transcrição/metabolismo , Western Blotting , Movimento Celular , Proliferação de Células , Células Cultivadas , Proteínas de Homeodomínio/genética , Humanos , Queratinócitos/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/metabolismo , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Well-characterized mice models will afford a cheaper, easy-handling opportunity for a more comprehensive understanding of 2009 influenza A (H1N1) virus's pathogenesis potential. We aimed to provide a robust description of pulmonary immune responses in the mice infected by the virus. METHODS: BALB/c mice were inoculated intranasally with A/Beijing/501/2009(H1N1) (BJ501) and A/PR/8/34(H1N1) (PR8) viruses and compared for survival rate, viral replication, and kinetics of pulmonary immune responses. RESULTS: BJ501 virus replicated less efficiently in the lungs than PR8, and both caused lethal illness in the mice. The transient increases of pulmonary TNF-α 2 days post infection for BJ501 and of INF-γ and IL-10 at 6 days post infection for PR8 were observed. IL-2+ and IL-4+ secreting cells showed significant increase 12 days post infection, while IFN-γ+, IgG+ and IgA+ secreting cells increased 6 days post infection. The different patterns of pulmonary immunological parameters between two viruses were at most seen in IL-6, IL-17 secretion and IgG1/IgG2a ratio. CONCLUSIONS: The BALB/c mouse is evaluated as a good pathogenic model for studying BJ501 2009 H1N1 virus. The work provided some basic and detailed data, which might be referred when further evaluating innate and adapted pulmonary immune responses and local viral load in mice.
Assuntos
Vírus da Influenza A Subtipo H1N1/imunologia , Infecções por Orthomyxoviridae/imunologia , Animais , Citocinas/análise , Feminino , Vírus da Influenza A Subtipo H1N1/química , Pulmão/química , Camundongos , Camundongos Endogâmicos BALB CRESUMO
It was found that Hg(2+) can inhibit the peroxidase mimetic activity of bovine serum albumin (BSA) protected Au clusters (BSA-Au) due to the specific interaction between Hg(2+) and Au(+) existed onto the surface of BSA-Au clusters. By coupling with 3, 3', 5, 5'-tetramethylbenzidine (TMB)-H2O2 chromogenic reaction, a novel method for Hg(2+) detection was developed based on the inhibiting effect of Hg(2+) on BSA-Au clusters peroxidase-like activity. This method exhibited high selectivity and sensitivity. As low as 3 nM (0.6 ppb, 3σ) Hg(2+) could be detected with a linear range from 10 nM (2 ppb) to 10 µM (2 ppm) and this method was successfully applied for the determination of total mercury content in skin lightening products.
Assuntos
Ouro/química , Mercúrio/análise , Nanopartículas Metálicas/química , Soroalbumina Bovina/química , Preparações Clareadoras de Pele/química , Benzidinas/química , Materiais Biomiméticos , Contaminação de Medicamentos , Humanos , Peróxido de Hidrogênio/química , Oxirredução , Peroxidase/química , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: In order to explore the regulating effects of Egr-1 promoter sequences in transcriptional targeting by 5-fluorouracil (5-Fu) on the expression of hematopoietic growth factor genes. METHODS: The human GM-CSF cDNA and enhanced green fluorescent protein (EGFP) cDNA were linked together with IRES and then inserted into the expression vector pCIneo under control of the Egr-1 promoter (Egr-EG). The vector was transferred into human bone marrow stromal cell line HFCL by lipofectin(TM). The transfected cell clones (HFCL/EG) have been selected by the addition of G418. The cells are exposed to the clinically important anticancer agent 5-fluorouracil. The activity of EGFP in HFCL/EG cells was detected by flow cytometry. The post-chemotherapeutical expression of GM-CSF in HFCL/EG was confirmed with ELISA and Western blot and RT-PCR respectively. The effect of N-acetylcysteine (a free radical scavenger) on GM-CSF production post-exposure to 5-Fu was examined. The HFCL/EG cells were transplanted intravenously into B16 melanoma in C. B-17 combined immunodeficient (SCID) mice. 5-Fu was given i.p. at Day 3. The white blood cell number in peripheral blood, the expression of EGFP and GM-CSF and in human stromal cell engrafted in recipient mice were detected by flow cytometry and RT-PCR respectively. Tumor volume in tumor-bearing mice was calculated. RESULTS: The results indicated that the activity of EGFP and the amounts of secreted GM-CSF in HFCL/EG cells exposed to 5-Fu increased as compared to non-5-Fu group with flow cytometry, RT-PCR and ELISA respectively. N-acetylcysteine significantly decreased the concentration of GM-CSF in HFCL/EG cells treated with 5-FU. In contrast to two control groups, HFCL/EG (Egr-1 regulatory element-derived expression of GM-CSF gene therapy) resulted in a proportionally obvious increase in the number of white blood cell after chemotherapy and no significant difference was found for tumor inhibition in recipient mice. CONCLUSIONS: These in vitro data provide an experimental basis for use of gene therapy of hematopoietic growth factor gene regulated by Egr-1 promoter to protect hematopoiesis from 5-Fu-injury effects.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/genética , Fluoruracila/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Hematopoese , Regiões Promotoras Genéticas , Animais , Células da Medula Óssea/efeitos dos fármacos , Linhagem Celular , Feminino , Expressão Gênica , Vetores Genéticos , Humanos , Camundongos , Camundongos SCID , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
BACKGROUND: H3N2 subtype influenza A viruses have been identified in humans worldwide, raising concerns about their pandemic potential and prompting the development of candidate vaccines to protect humans against this subtype of influenza A virus. The aim of this study was to establish a system for rescuing of a cold-adapted high-yielding H3N2 subtype human influenza virus by reverse genetics. METHODS: In order to generate better and safer vaccine candidate viruses, a cold-adapted high yielding reassortant H3N2 influenza A virus was genetically constructed by reverse genetics and was designated as rgAA-H3N2. The rgAA-H3N2 virus contained HA and NA genes from an epidemic strain A/Wisconsin/67/2005 (H3N2) in a background of internal genes derived from the master donor viruses (MDV), cold-adapted (ca), temperature sensitive (ts), live attenuated influenza virus strain A/Ann Arbor/6/60 (MDV-A). RESULTS: In this presentation, the virus HA titer of rgAA-H3N2 in the allantoic fluid from infected embryonated eggs was as high as 1:1024. A fluorescent focus assay (FFU) was performed 24-36 hours post-infection using a specific antibody and bright staining was used for determining the virus titer. The allantoic fluid containing the recovered influenza virus was analyzed in a hemagglutination inhibition (HI) test and the specific inhibition was found. CONCLUSION: The results mentioned above demonstrated that cold-adapted, attenuated reassortant H3N2 subtype influenza A virus was successfully generated, which laid a good foundation for the further related research.
Assuntos
Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza/imunologia , Animais , Células COS , Chlorocebus aethiops , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Camundongos , Camundongos Endogâmicos BALB C , Neuraminidase/genética , Plasmídeos , Vírus Reordenados/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vacinas Atenuadas/imunologia , Proteínas Virais/genéticaRESUMO
In order to explore the regulatory effects of Egr-1 promoter sequences induced by doxorubicin (ADM) in transcriptional targeting on the expression of hematopoietic growth factor genes. The human GM-CSF cDNA and enhanced green fluorescent protein (eGFP) cDNA were linked together with internal ribozyme entry site (IRES) and then inserted into the expression vector pCIneo under control of the Egr-1 promoter (Egr-EG). The vector was transferred into human bone marrow stromal cell line HFCL by lipofectin. The transfected cell clones (HFCL/EG) were selected by the addition of G418. The cells were exposed to the clinically important anticancer agent doxorubicin. The activity of eGFP in HFCL/EG cells was detected by flow cytometry. The amounts of GM-CSF in HFCL/EG postchemotherapy were confirmed with ELISA. The effect of GM-CSF in HFCL/EG cultural supernatants on expansion of CFU-GM derived from cord blood was also studied. The effect of N-acetylcysteine (a free radical scavenger) on GM-CSF production following exposure to ADM was examined. The results indicated that the activity of eGFP and the amounts of secreted GM-CSF in HFCL/EG cells exposed to ADM increased as compared to non-ADM group. The effect of GM-CSF in HFCL/EG cultural supernatants on expansion of CFU-GM was significantly higher than that of non-ADM group. N-acetylcysteine significantly decreased the concentration of GM-CSF produced by HFCL/EG treated with ADM. It is concluded that these in vitro data provide an experimental basis for the use of gene therapy of hematopoietic growth factor gene regulated by Egr-1 promoter to protect hematopoiesis from ADM-injury.
Assuntos
Doxorrubicina/farmacologia , Proteína 1 de Resposta de Crescimento Precoce/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Sequência de Bases , Células da Medula Óssea/citologia , Linhagem Celular , Regulação da Expressão Gênica , Vetores Genéticos , Humanos , Regiões Promotoras GenéticasRESUMO
AIM: To express the HSP65-MUC1 VNTR(2) in E.coli and to evaluate its activity of inhibiting tumor growth in vivo. METHODS: HSP65 and MUC1 VNTR(2) were generated by PCR method and sub-cloned to pET28a(+) to construct the recombinant expression vector HSP65-MUC1 VNTR(2)-pET28a(+). E.coli BL21(DE3) bearing the plasmid was induced with IPTG for protein production. Target protein was characterized by Western blot with monoclonal antibody and purified by Q-Sepharose ion-exchange chromatography and gel filtration. The murine cancer cell linejB16 that transfected by human gene MUC1 was utilized to construct the model of carcinoma, and the tumor growth inhibition activities of HSP65-MUC1VNTR(2) was evaluated in mice C57BL/6. RESULTS: The gene HSP65 and MUC1 VNTR(2) confirmed by sequence analysis matched respectively with BCG HSP65 and human gene MUC1 VNTRs in GenBank exactly. The reconstructed vector HSP65-MUC1 VNTR(2)-pET28a could express target protein stably in the soluble fraction of bacterial extract. The purity of HSP65-MUC1 VNTR(2) protein could be above 95% after purification by Q ion-exchange chromatography and gel filtration. The result of Western blot with monoclonal antibody showed positive. The results of prophylactic immunization with HSP65-MUC1 VNTR(2) fusion protein showed that experiment all groups had significantly higher tumor inhibition rates than that of control group. CONCLUSION: In summary, HSP65-MUC1 VNTR(2) fusion protein was solubly expressed in prokaryotic expression system and its tumor growth inhibition activity was evaluated primarily. The result indicated that the fusion protein could inhibit the MUC1 positive tumor growth significantly. It can be used in the future research as the cancer vaccine.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Chaperoninas/genética , Chaperoninas/farmacologia , Repetições Minissatélites/genética , Mucina-1/genética , Mucina-1/farmacologia , Mycobacterium bovis , Animais , Antineoplásicos/isolamento & purificação , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Chaperonina 60 , Chaperoninas/biossíntese , Chaperoninas/isolamento & purificação , Escherichia coli/genética , Humanos , Camundongos , Mucina-1/biossíntese , Mucina-1/isolamento & purificação , Células Procarióticas/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
OBJECTIVE: To evaluate the effect of Long-term exposure to low intensity microwave radiation on male reproductivity. METHODS: A total of 289 married male radar operators were included in the radar group and 148 married men unexposed to microwave radiation were enrolled as controls. Questionnaires were used and the intensity of microwave radiation in different working areas was detected. RESULTS: The rate of sexual dysfunction was 43.6% in the radar group and 24.4% in the control group (P < 0.01). The natural pregnancy rate was 53.6% within 1 year of marriage and 46.4% after 1 year of marriage in the radar group, as compared with 81.1% and 18.9% in the control group (P < 0.01). CONCLUSION: Long-term exposure to low intensity microwave radiation evidently increased the sexual dysfunction rate and decreased natural pregnancy rate in men.
Assuntos
Disfunção Erétil/epidemiologia , Fertilidade/efeitos da radiação , Micro-Ondas , Relação Dose-Resposta à Radiação , Feminino , Humanos , Masculino , Micro-Ondas/efeitos adversos , Militares , Exposição Ocupacional , Gravidez , Resultado da Gravidez , Radar , Doses de Radiação , Inquéritos e QuestionáriosRESUMO
OBJECTIVE: To investigate the semen quality of the Chinese army men. METHODS: Ten-item sperm quality analyses were made by manual methods and the computer assisted sperm analysis system in 1054 young Chinese army men. The subjects were divided into 4 age groups (18-20 yrs., 21-25 yrs., 26-30 yrs and 31-35 yrs.), and the results of the analyses were compared. RESULTS: Among the 1 054 young males investigated, the semen volume was (2.6 +/- 1.4) ml, sperm density (55.9 +/- 46.5) x 10(6)/ml, sperm grade a + b motility (47.1 +/- 19.0)%, sperm viability (70.6 +/- 22.1)%, morphologically normal sperm (84.7 +/- 10.2)%, and acrosomal integrity (86.1 +/- 7.2)%. As for the percentages of the quality indexes that met WHO standards, the sperm volume was 73.5%, liquefaction time 91.1%, pH 93.0%, grade a + b motility 45.5%, viability 86.7%, sperm density 80.4%, morphologically normal sperm 98.2%, and the sperm total number 78.0%. Those who accorded with all the WHO standards accounted for 40.2%. CONCLUSION: The semen quality of the 18-35 year old army men was better than previously reported in the similar literature. And that of the 26-30 yrs. group was the best among all the age groups.