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

OBJECTIVETo study the expression and localization of early growth response gene-1 (Egr-1) in macrophages after stimulation by silicon dioxide in vivo and in vitro and to discuss the role of Egr-1 in the development of silicosis.

METHODSThe expression of Egr-1 in animal model of silicosis was analyzed by using immunohistochemistry. Western-blot, immunofluorescence and RT-PCR analysis were used to detect the expression and localization of Egr-1 protein and the dynamic changes of Egr-1 mRNA in cultured macrophages RAW264.7, after stimulation by silicon dioxide.

RESULTSIn animal model with induced silicosis, there was an increased expression of Egr-1 in pulmonary macrophages. The expression levels peaked at the 14th day. In vitro, the transcription of Egr-1 increased in RAW264.7 macrophages during 15 to 240 minutes after the administration of silicon dioxide. The response peaked at 15 minutes and diminished to a minimal level at 480 minutes. Nuclear translocation was most apparent at 60 minutes, lasted till 120 minutes and diminished gradually. During the period from 60 to 120 minutes, the expression of Egr-1 protein also reached a peak.

CONCLUSIONSSilicon dioxide can activate the nuclear transcription factor Egr-1 in vivo and in vitro in macrophages. Egr-1 may thus play an important pathogenetic role in the development of silicosis.

Animals ; Blotting, Western ; Cell Line ; DNA-Binding Proteins ; genetics ; metabolism ; Early Growth Response Protein 1 ; Gene Expression Regulation ; drug effects ; Immediate-Early Proteins ; Immunohistochemistry ; Lung ; drug effects ; metabolism ; pathology ; Macrophages ; drug effects ; metabolism ; Macrophages, Alveolar ; drug effects ; metabolism ; pathology ; Male ; RNA, Messenger ; drug effects ; genetics ; metabolism ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; Silicon Dioxide ; pharmacology ; Transcription Factors ; genetics ; metabolism

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 discuss the role of early growth response factor (Egr)-1 and it's upstream signaling pathway in the development of silicosis.

METHODSThe expression and localization of Egr-1 were analyzed by immunofluorescence and in-situ hybridization. The activity of Egr-1 was observed in treated cells by using a reporter plasmid and EMSA, the activity of ERK1/2 in RAW264.7 incubated with SiO(2) by using a kinase assay, and further by using a kinase inhibitor assay to investigate the role of upstream kinase in the signal pathway of the activation of Egr-1.

RESULTSThe obvious increase of expression and transcription of Egr-1 was observed shortly after being treated by silica and its activity increased abruptly. There was an increase of the activity of ERK1/2 in RAW264.7 cells treated, which reached a peak at 30 minutes. The expression and transcription of Egr-1 decreased maniferstly after using kinase inhibitors.

CONCLUSIONEgr-1 expression can be induced by silica dioxide in RAW264.7 cells, and the ERK1/2, p38 kinases may take part in this process which suggest the pathway of SiO(2), ERK1/2, p38 and Egr-1 may play an important role in the development of silicosis.

Animals ; Butadienes ; pharmacology ; Cells, Cultured ; Early Growth Response Protein 1 ; biosynthesis ; genetics ; physiology ; Enzyme Inhibitors ; pharmacology ; Gene Expression Regulation ; Macrophages ; metabolism ; Mice ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Nitriles ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Signal Transduction ; Silicon Dioxide ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; metabolism

AIMTo study the effect of Safflower injection (a compound of Chinese Traditional medicine) on pulmonary hypertension in rat during chronic hypoxia and hypercapnia.

METHODSSprague-Dawley rats were randomly divided into normal control group (A), hypoxic hypercapnic group (B), hypoxic hypercapnia + Safflower injection group (C). The concentration of TXB2 and 6-keto-PGF18 in plasma and in lung homogenate were detected by the radioimmunoassay.

RESULTS(1) mPAP, weight ratio of right ventricle (RV) to left ventricle plus septum (LV + S) were much higher in rats of hypoxic hypercapnic group than those of control group. Differences of mCAP among the three groups were not significant. (2) The concentration of TXB2 and the ratio of TXB2/6-keto-PGF1a were significantly higher in rats of B group than those of A and C group. (3) The results examined by light microscopy showed that WA/TA (vessel wall area/total area), SMC (the density of medial smooth muscle cell) and PAMT (the thickness of medial smooth cell layer) were significantly higher in rats of B group than those of A and C group. (4) The results examined by electron microscopy showed proliferation of medial smooth muscle cells and collagen fibers of pulmonary arterioles in rats of B group, and Safflower injection could reverse the changes mentioned above.

CONCLUSIONSafflower injection may inhibit hypoxic hypercapnia pulmonary hypertension and pulmonary vessel remodeling by decreasing the ratio of TXB2/6-keto-PGF1a.

6-Ketoprostaglandin F1 alpha ; metabolism ; Animals ; Carthamus tinctorius ; chemistry ; Drugs, Chinese Herbal ; pharmacology ; Hypercapnia ; metabolism ; pathology ; physiopathology ; Hypertension, Pulmonary ; prevention & control ; Hypoxia ; metabolism ; pathology ; physiopathology ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Thromboxane B2 ; metabolism

Objective To evaluate the preventive and therapeutic effect of oral solution of Niao Du Kang (尿毒康) on acute renal tubular necrosis (ATN) in Sprague-Dawley (SD) rats,and preliminarily approach its mechanisms.Methods The oral solution was composed of traditional Chinese medicinal herbs of Radix et Rhizoma Rhei (大黄),Radix Sanguisorbae Offieinalis (地榆),Radix Salviae Miltiorrhizae (丹参), Radix Astragali seu Hedysari (黄芪),Flos Carthami Tinctorii (红花) and so on.Sixty-four SD male rats were randomly divided into 4 groups:normal control group,ATN model group,verapamil treatment group and traditional Chinese medicine,oral solution of Niao Du Kang treatment group,every group having 16 rats.. The ATN model of SD rats was induced by intramuscular injection of 50% glycerin mixed with isotonic saline solution at the back of bilateral lower extremities.After the model was established in each group ,the levels of blood urea nitrogen (BUN),levels of blood serum creatinine (SCr),renal failure index (RFI) and renal pathological changes were detected at different time points after modeling for 12 and 24 hours,and the therapeutic effect of oral solution of Niao Du Kang was observed.Results After the model establishment, Niao Du Kang oral solution could lower the elevation of blood BUN,SCr,RFI in the model,and compared with the normal control group,there were statistical significances (all P

Objective To detect EIA factor expression in human rectal cancer and normal tissue and to determine whether it is correlated with invasion and metastasis of human rectal cancer. Methods Real- time RT-PCR was used to detect E1AF expression in matched rectal cancers and normal tissues from g6 in- patients.Results Among the 86 rectal cancer samples tested,55 cases E1AF mRNA overexpression was ob- served. The mRNA expression of E1AF in the sample group was remarkably different from that in the control group.In carcinomas,E1AF mRNA expression correlated significantly with histological type,depth of inva- sion,lymph node and distant metastasis and advanced Duke stage.Conclusion E1AF is correlated signifi- cantly with invasion and metastasis of human rectal cancer and may be an important factor in the invasion and metastasis.

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