This study aimed to construct recombinant adenovirus expressing Epstein-Barr nuclear antigen 1 (EBNA1) against nasopharyngeal carcinoma (NPC). The C-terminal region fragment of the ebna1 gene of Epstein-Barr virus was amplified from the standard strain B95-8 by polymerase chain reaction (PCR). The gene fragment was inserted into the pDC316 shuttle plasmid using the EcoRI and BgIII restriction enzyme sites. The pDC316-ebna1 shuttle plasmid and pBHG helper plasmid were cotransfected into HEK293 cells after sequencing. The soluble protein was extracted from HEK293 cells, which caused apparent cytopathic effects. The transcription and expression of the ebna1 gene were confirmed using flow cytometry and Western blotting. rAd-ebna1 titers were measured by the TCID50. rAd-ebna1 was injected into BALB/c mice at a dose of 2 x 10(8) VP per mouse, EBNA1 epitope-specific responses were measured at 1st, 2nd, 4th and 8th weeks post-immunization. The target fragment of ebna1 (939 bp) was obtained by PCR, and was in consensus with the sequence from the standard strain B95-8. Cytopathic effects were observed after the pDC316-ebna1 shuttle plasmid and pBHG helper plasmid were cotransfected into HEK293 cells. rAd-ebna1 was successfully recombined in HEK293 cells. EBNA1 protein was detected in HEK293 cells, rAd-ebna1 titers reached 10(8) TCID50/mL. Specific responses to CD4+ epitopes of EBNA1 were detected in the immunized mice. In conclusion, rAd-ebna1 was successfully constructed and induced specific responses to CD4+ epitopes of EBNA1 in immunized mice.
Adenoviridae ; genetics ; immunology ; Animals ; CD4-Positive T-Lymphocytes ; immunology ; virology ; Epstein-Barr Virus Infections ; immunology ; prevention & control ; virology ; Epstein-Barr Virus Nuclear Antigens ; administration & dosage ; genetics ; immunology ; Genetic Vectors ; genetics ; immunology ; Herpesvirus 4, Human ; genetics ; immunology ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Viral Proteins ; administration & dosage ; genetics ; immunology

This article studied the chemical constituents from the aerial part of Vitis thunbergii var. taiwaniana. The 60% ethanol extract was eluted with 95% ethanol though HP-20 macroporous adsorption resin column. 12 compounds, including (1) betulinic acid, (2)2, 2, 2'-bis (4-hydroxyphenyl) propane bis (2, 3-epoxypropyl) ether, (3) eriodictyol, (4) trans-ε-viniferin, (5) (+)-cis-ε-viniferin, (6) kobophenol A, (7) ampelopsin A, (8) nepalensinol B, (9) cis-miyabenol C, (10) cis-vitisin B, (11) cis-gnetin H and (12) (+)-hopeaphenol, were separated by using normal phase silica gel, ODS, Sephdadex LH-20 column chromatographies and semi-preparative or preparative HPLC. Compounds 2, 5, 6, 8, 9, 10, 11 were separated from the genus Vitis for the first time and compounds 3, 7, 12 were separated from Vitis thunbergii var. taiwaniana for the first time. At a concentration of 50 μmol · L(-1), compound 6, 7 and 11 showed strong cytotoxicity against MCF-7 cell lines with the inhibition rate of 66.58%, 57.16%, 52.84%, respectively.
Antineoplastic Agents, Phytogenic ; pharmacology ; Humans ; MCF-7 Cells ; Plant Extracts ; analysis ; pharmacology ; Vitis ; chemistry

This study was aimed to clone the gene coding mouse fibroblast growth factor receptor-1 (fgfr1), to construct the recombinant lentiviral vector of truncated form fgfr-1 (Δfgfr1) carrying enhanced green fluorescence protein (EGFP) and to investigate its expression in eukaryotic cells (293FT cells). The full length fgfr1 gene was cloned by RT-PCR using brain tissue of BALB/c fetal mouse as template and inserted into PCR-Blunt vector, a truncated fgfr1 fragment was produced by site-directed mutagenesis for deleting intracellular phosphorylated domain, then was subcloned into a lentiviral vector and cotransfected into 293FT packaging cells together with envelope plasmid and packaging plasmid by lipofectamine 2000. Viruses were gathered and concentrated using ultracentrifuge, and then transfected into 293FT cells. Expression of EGFP was detected by fluorescent microscopy and flow cytometry (FCM), and the truncated FGFR1 protein was detected by Western blot. The results demonstrated that mouse fgfr1 gene was cloned and the lentiviral expression vector LV-IRES-EGFP-Δfgfr1 and control vector LV-IRES-EGFP were successfully constructed. The lentiviral particles were correctly packaged, and the virus titers were above 10(8) TU/ml in the supernatant after concentration. Expression of EGFP was detected by fluorescent microscopy in 293FT cells post transfection, and the transfection efficacy was > 95% determined by FCM. Expression of FGFR1 protein detected by Western blot was significantly higher than that in control group. It is concluded that the truncated gene fgfr1 along with the gene coding EGFP is successfully inserted into a lentiviral vector to construct a recombinant lentiviral vector, which can be expressed in eukaryotic cells.
Animals ; Cell Line ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Mice ; Mice, Inbred BALB C ; Plasmids ; Receptor, Fibroblast Growth Factor, Type 1 ; genetics ; Recombinant Fusion Proteins ; genetics ; Transfection

Standardized Manipulation of Acupuncture and Moxibustion Part 8: Intradermal Needle was compiled with the following principles. The compiling standard, technical features and clinic manipulations of intradermal needle were taken as the basic principle for compiling. Literature research, expert survey and clinic practice verification were applied as the drafting methods. The key issues were focused on the relationship between standardization and individualization, normalization and effectiveness, qualification and quantification. And the postural selection, reinforcing and reducing manipulations, fixing materials and embedding duration involved in intradermal needling were emphasized particularly. At the same time, details and the future way of thinking of intradermal needle were expounded in this article as well.
Acupuncture Therapy ; instrumentation ; standards ; China ; Humans ; Moxibustion ; standards ; Needles ; standards ; Reference Standards

OBJECTIVETo study the effect of celecoxib on chronic hypoxia and hypercapnic pulmonary hypertension.

METHODSSD rats were randomly divided into normal control group (A), hypoxic hypercapnic group (B), hypoxic hypercapnia+ celecoxib group (C). The content of TXB2 and 6-keto-PGF1alpha in plasma and lung were detected by the technique of radioimmunology.

RESULTS(1) Mean pulmonary arteria pressure(mPAP) was significantly higher in rats of B group than those of A group. mPAP was significantly higher in rats of C group than those of B group. Differences of mPAP were not significant in three groups. (2) The content of TXB2 in plasma and lung and the ratio of TXB2/6-keto-PGF1alpha were significantly higher in rats of B group than those of A group. The ratio of TXB2/6-keto-PGF1alpha was significantly higher and the content of 6-keto-PGF1alpha in plasma and lung was significantly lower in rats of C group than those of B group. (3) Light microscopy showed that WA/TA (vessel wall area/total area) and PAMT (the thickness of medial smooth cell layer) were significantly higher in rats of B group than those of A group. WA/TA and PAMT were significantly higher in rats of C group than those of B group. (4) Electron microscopy showed the thickening of vessel wall and the proliferation of collagen fiber in B group and augmentation of smooth muscle cell and abundance of myofilament in pulmonary arterioles in C group.

CONCLUSIONCelecoxib can aggravate hypoxic hypercapnia pulmonary hypertension and pulmonary vessel remodeling by increasing the ratio of TXA2/PGI2.

Animals ; Celecoxib ; Chronic Disease ; Cyclooxygenase 2 Inhibitors ; adverse effects ; pharmacology ; Epoprostenol ; blood ; Hypercapnia ; complications ; Hypertension, Pulmonary ; etiology ; physiopathology ; Hypoxia ; complications ; Male ; Pyrazoles ; adverse effects ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Sulfonamides ; adverse effects ; pharmacology ; Thromboxane A2 ; blood

OBJECTIVETo study the correlation between the expression of Egr-1 and NF-kappaB and the up-regulation of TNF-alpha and TGF-beta1 in macrophages after stimulation by silica in-vitro.

METHODSMacrophages were treated with antibodies against Egr-1 and NF-kappaB and antisense oligonucleotides. The level of TNF-alpha protein in the cell supernatant was then measured using enzyme-linked immunoadsorbent assay (ELISA). The expression of TGF-beta1 protein was detected by immunocytochemistry. The expression of TNF-alpha and TGF-beta1 mRNAs was also monitored by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTSCompared with silica-stimulated macrophages untreated with antibodies, the cells treated with 10 micro g/ml of Egr-1 or NF-kappaB antibodies were associated with reduced expression of TNF-alpha and TGF-beta1 proteins and mRNAs (P < 0.05). Compared with silica-stimulated untransfected group, the antisense group was associated with obvious reduction in the expression of TNF-alpha and TGF-beta1 proteins and mRNAs (P < 0.05).

CONCLUSIONThe expression of TNF-alpha and TGF-beta1 mRNAs and proteins are associated with activation of Egr-1 and NF-kappaB in macrophages, after stimulation by silica. It is possible that the corresponding antibodies and antisense oligonucleotides may become a potential therapeutic tool in the management of silicosis in the future.

Animals ; Antibodies ; immunology ; Cells, Cultured ; DNA-Binding Proteins ; genetics ; immunology ; Early Growth Response Protein 1 ; Immediate-Early Proteins ; genetics ; immunology ; Macrophages ; cytology ; metabolism ; Mice ; NF-kappa B ; genetics ; immunology ; Oligonucleotides, Antisense ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Silicon Dioxide ; pharmacology ; Silicosis ; etiology ; Transcription Factors ; genetics ; immunology ; Transforming Growth Factor beta ; biosynthesis ; genetics ; Transforming Growth Factor beta1 ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics

OBJECTIVETo explore the mechanism of occupational medicamentosa-like dermatitis (OMDT) induced by trichloroethylene (TCE) and some immunity indexes in workers occupationally exposed to TCE.

METHODSThe blood samples from 8 cases with medicamentosa-like dermatitis in 1st, 2nd, 3rd, 4th and 5th weeks after admitting to hospital were examined for liver function, immunoglobulin and some complement indexes. Thirty nine workers occupationally exposed to TCE were investigated for urinary TCE and some immuno-complement indexes. The TCE concentrations of air in workplaces were monitored.

RESULTSC3d-CIC and C3 of patients before admission were (92.86 ± 44.80) mg/L and 0.91 ± 0.19 mg/L, respectively. C3d-CIC and C3 of patients before discharge were (52.41 ± 17.75) mg/L and (1.14 ± 0.22) mg/L, respectively. There were significant differences between admission and discharge (P < 0.05). The average TCE concentration in 4 workplaces was (351.96 ± 36.72) mg/m(3), which was higher than the occupational exposure limits (OELs). The number of workers exposed to the TCE concentration-time weighted and TCA in urine over OELs were 28.21% and 56.41% of total subjects, respectively. The serum IgG and CIC levels of patients before discharge were (10.03 ± 1.21) mg/L and 103.50 ± 29.17 mU/L, which were significantly lower than those (17.21 ± 1.85) mg/L and (227.46 ± 111.67) mU/L of patients before admission (P < 0.01).

CONCLUSIONThe type II and III hypersensitivity may be associated with OMDT and the organ injure induced by TCE.

Adolescent ; Adult ; Complement System Proteins ; immunology ; Dermatitis, Occupational ; immunology ; Female ; Humans ; Male ; Occupational Exposure ; Trichloroethylene ; toxicity ; Young Adult

OBJECTIVETo study the role of Th17 cells in acute intestine graft-versus-host disease following allogenetic bone marrow transplantation(allo-BMT).

METHODSMice were split randomly into five groups: normal control, irradiated, allo-BMT, allo-BMT + DMSO and allo-BMT + Halofuginone (HF) groups. HF was given intraperitoneally at a dose of 5 µg per mouse from -1 d to 10 d after allo-BMT. aGVHD symptoms were followed-up to perform clinical and pathogenic scores. The levels of Th1/Th17, interleukin-17 and interferon-γ were measured by flow cytometry at day 7 d. mRNA expressions of T-bet, RORγT, CXCL9, CXCL10, CXCL11 and CCL20 in intestine were evaluated by real-time PCR.

RESULTSIntestinal damages in allo-BMT-HF mice was more serious than in normal control and allo-BMT groups at day 14 after transplantation. At day 7, Th17 ratio in allo-BMT + HF group was significantly lower than in allo-BMT group. IL-17A was not detected, but Th1 ratio was higher in allo-BMT + HF. There was a similar increment in the relative expressions of T-bet in both allo-BMT and allo-BMT + HF groups. Expressions of CXCL9 and CXCL10 elevated in allo-BMT + HF group, which were significantly higher than those in allo-BMT group (P < 0.01). CCL20 expression significantly increased in allo-BMT group, but it was not detected in allo-BMT + HF group.

CONCLUSIONBlockage of th17 cells differentiation exacerbated acute intestine graft versus-host disease.

Animals ; Bone Marrow Transplantation ; adverse effects ; Cell Differentiation ; Female ; Graft vs Host Disease ; pathology ; Intestinal Diseases ; pathology ; Intestines ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Th17 Cells ; cytology ; Transplantation, Homologous

OBJECTIVETo reseach the correlations between cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) expressions and angiogenesis in pharyngeal tissue of patients with obstructive sleep apnea hypopnea syndrome (OSAHS).

METHODSBiopsies were obtained by uvulopalatopharyngoplasty from 40 patients with mild to severe OSAHS. Control specimens of palatopharyngeal and palatoglossal arch mucosa were retreved from 6 patients with chronic tonsillitis and proved have no related disorders. HE was used to observe the changes of pharyngeal tissue, immunohistochemical staining with antibodies against COX-2, VEGF, microvessel density (MVD) (marked with CD34).

RESULTSCOX-2 and VEGF mainly expressed at pavement-epithelium and glandular epithelium of pharyngeal tissue, and stronger COX-2 and VEGF expression was found in midrange and severe OSAHS than mild and control group (P < 0.01), so as MVD. COX-2 expression was correlated positively with VEGF expression, and had significant correlation with MVD. VEGF expression had the same correlation with MVD. These three targets had considerable relation with apnea hypopnea index (AHI) and lowest O2 saturation at night.

CONCLUSIONThere was angiogenesis which had important relationship with hypoxia degree in patients of OSAHS, and COX-2 and VEGF play a crucial role in its development.

Adult ; Cyclooxygenase 2 ; genetics ; metabolism ; Female ; Humans ; Male ; Middle Aged ; Neovascularization, Pathologic ; Pharynx ; blood supply ; metabolism ; Sleep Apnea, Obstructive ; metabolism ; physiopathology ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

OBJECTIVETo prepare the genetic engineering regulatory T cells (Treg) via the self-inactivating (SIN) lentiviral vectors carrying Foxp3 gene, and assay the phenotype and abilities of its proliferation and immunosuppression.

METHODSThe bicistronic SIN lentiviral transfer plasmid containing Foxp3 gene and internal ribosomal entry site-green fluorescent protein gene (IRES-GFP) was constructed. Human embryonic kidney 293T cells were co-transfected using liposome by lentiviral packing system, which included the packaging plasmid Delta NRF, the transfer plasmid and the envelope plasmid VSVG. The efficiency of gene transduction and the expressions of Foxp3, CD25, GITR, CTLA-4 of CD4(+)CD25(-) T cells, which were isolated by magnetic beads from the spleen, and then co-cultured with 293T cells, were detected by flow cytometry (FCM). The proliferative and suppressive capacities of transduced T cells were estimated by Cell Count Kit-8 (CCK-8) and the cytokine production was performed by ELISA.

RESULTSThe lentiviral transfer plasmid pXZ208-Foxp3-IRES-GFP was successfully constructed, the virus titers were above 10(6) IU/ml in the supernatant. pXZ208-IRES-GFP was used as control group. After cocultured, the CD4(+)CD25(-) T cells expressed significantly higher Foxp3, CD25, GITR and CTLA-4 in experimental group than in control group. Upon stimulation with anti-CD3 epsilon and APCs, the proliferative capacity of Foxp3-transduced T cells and the production of IL-2, IL-4, IL-10, IFN-gamma were significantly lower than those in control group (P < 0.01); Foxp3-transduced T cells also significantly inhibited the proliferation of CD4(+)CD25(-) T cells.

CONCLUSIONSThe genetic engineering Treg mediated by SIN lentiviral vectors are successfully constructed and their phenotype and function are similar to natural CD4(+)CD25(+) Treg.

Animals ; Cell Proliferation ; Cells, Cultured ; Forkhead Transcription Factors ; genetics ; metabolism ; Genetic Engineering ; Genetic Vectors ; HEK293 Cells ; Humans ; Lentivirus ; genetics ; Mice ; Mice, Inbred BALB C ; Phenotype ; T-Lymphocytes, Regulatory ; cytology ; immunology ; metabolism ; Transfection