HER2, a member of epidermal growth factor receptor family proteins, is overexpressed in about 30% of human breast cancer. Increased levels of HER2 are associated with poor patient prognosis and enhanced metastasis. RNA interference (RNAi) is developed recently as a new technique which can inhibit gene expression specifically in mammalian cells. On the basis of previous study,in which two target sequences with favorable RNAi effect on HER2 were identified, a series of dual promoter siRNA-expressing vectors containing two opposing U6 and H1 promoters were constructed. After transfection of HER2-overexpressing SKBR3 breast cancer cells with the siRNA-expressing vectors, downregulation of HER2 was identified quantitatively. Subsequently, the siRNA-expressing cassettes were subcloned into lentiviral vectors by LR recombination reaction and lentivirus was prepared successfully. The results from infection of SKBR3 cells with siRNA-expressing lentivirus demonstrated that lentiviral-mediated RNAi could downregulate HER2 expression efficiently through fluorescent quantitative PCR (FQ-PCR), western blot, and FACS analysis. Furthermore, cell growth was inhibited in cell proliferation assay after treatment with siRNA lentivirus.A new tool for clarifying the function of HER2 in cancer metastasis and developing the gene therapy drug was offered.

Acute Disease ; Carbon Monoxide Poisoning ; enzymology ; physiopathology ; Creatine Kinase ; metabolism ; Creatine Kinase, MB Form ; Echocardiography ; Heart ; physiopathology ; Humans ; Hyperbaric Oxygenation ; Isoenzymes ; metabolism ; Myocardium ; enzymology

Processed Chinese medicine is the core of traditional Chinese medicine (TCM) industry chain,which directly affects the clinical efficacy and. safety of Chinese patent medicine and clinical formula Decoction pieces. Studied the variation of effective substance in vivo Chinese medicine processing before and after processed, clarifying the effective substance and processing principle is a top priority of the development of Chinese medicine processing. The traditional research method chiefly focus on the variation about chemicals in vitro of processed Chinese medicine, it cannot reveal that the integrity and complexity of processed Chinese medicine efficacy changes, so the change process is the focus of future research in vivo on the base of effective substance of TCM This paper described the research on the base of effective substance of TCM and Processed Chinese medicine research status in vitro, discussed the analytical methods (plasma chemistry, pharmacokinetics, metabonomics) of the dynamic process in vivo about processed Chinese medicine, and pointed out development and related problems in process in vivo on the base of effective substance of TCM, which could provided research ideas and methods for in-depth interpretation of Chinese medicine processing mechanism.
Animals ; Drugs, Chinese Herbal ; analysis ; pharmacokinetics ; Humans ; Medicine, Chinese Traditional

Gardeniae Fructus contains volatile ingredients, however, the species and proportions in different processed products of Gardeniae Fructus are different. In this experiment, volatile ingredients were separated by steam distillation with content of 1.2, 1.0, 0.9, 0.7 µL · g(-1) in Gardeniae Fructus, fried Gardeniae Fructus, stir-baked Gardeniae Fructus, Gardeniae Fructus fried into carbon respectively. One hundred and twenty-four kinds of volatile components were identified by GC-MS. Fifty-three kinds of volatile ingredients consisted in Gardeniae Fructus accounting for 93.85%, 54 kinds in fried Cardeniae Fructus accounting for 92.01%, 32 kinds in stir-baked Cardeniae Fructus accounting for 91.59% and 43 kinds in Gardeniae Fructus fried into carbon accounting for 90.81%. In this paper, analysis of Gardeniae Fructus by GC-MS provides a scientific basis for elucidating the mechanism of different processed products.
Chemistry, Pharmaceutical ; Drugs, Chinese Herbal ; chemistry ; Gardenia ; chemistry ; Gas Chromatography-Mass Spectrometry ; Molecular Structure ; Volatile Organic Compounds ; chemistry

To determine the chemical composition of Galla chinensis extract (GCE) by several analysis techniques and to compare the efficacy of GCE and its main component(s) in inhibition of enamel demineralization, for the development of future anticaries agents, main organic composition of GCE was qualitatively determined by liquid chromatography-time of flight-mass spectrometry (LC-TOF-MS) and quantified by high-performance liquid chromatography-diode array detector (HPLC-DAD). Inorganic ions were tested by inductively coupled plasma-atomic emission spectroscopy and F was especially measured by ion chromatography. Then, bovine enamel blocks were randomly divided into four treatment groups and were subjected to a pH-cycling regime for 12 times. Each cycle included 5-min applications with one of four treatments: 4 g⋅L(-1) GCE solution, 4 g⋅L(-1) gallic acid (GA) solution, 1 g⋅L(-1) NaF solution (positive control), deionized water (DDW, negative control), and then 60-min application in pH 5.0 acidic buffer and 5-min application in neutral buffer. Acidic buffers were retained for calcium analysis. The main organic composition of GCE were GA and its isomer, and, to a lesser extent, small molecule gallotannins. The content of GA in GCE was 71.3%±0.2% (w/w). Inorganic ions were present in various amounts, of which Ca was (136±2.82) µg⋅g(-1), and Zn was (6.8±0.1) µg⋅g(-1). No F was detected in GCE. In pH cycling, GA showed an effect similar to GCE in inhibiting enamel demineralization (P>0.05). GA was found to be the main effective, demineralization inhibiting component of GCE and could be a promising agent for the development of anticaries agents.
Animals ; Calcium ; analysis ; Cariostatic Agents ; therapeutic use ; Cattle ; Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Dental Enamel ; Drugs, Chinese Herbal ; chemistry ; therapeutic use ; Gallic Acid ; analysis ; therapeutic use ; Hydrolyzable Tannins ; analysis ; Mass Spectrometry ; Polyphenols ; analysis ; Random Allocation ; Tooth Demineralization ; prevention & control

Objective To evaluate the performance of diluted thrombin time (dTT) assay for detecting Dabigatran levels and observe whether this assay may meet the requirements of clinical laboratory.Methods According to EP15-A2,EP6-A,EP7-A and C-24 documents of the Clinical and Laboratory Standards Institute (CLSI),the precision,trueness,analytical measurement range,carryover rate and anti-biological interference of dTT assay were evaluated and the stability of specimen for dTT assay was observed.Results Both the within-day and between-day coefficient of variation (CV) of dTT assay for detecting Dabigatran levels were consistent with manufacturer's stated CV.Compared with target values of Dabigatran,the relative bias of 3 levels of proficiency test materials from College of American Pathologists (CAP) were less than 10％.The results meet linear verification when Dabigatran concentration was between 30.92 and 249.13 ng/mL.The carryover rate was-0.84％.There was no interference for Dabigatran levels by dTT assay for detecting Dabigatran when Hb≤3 g/L,triglyceride≤873 mg/dL,heparin≤2.2 IU/mL and FDP≤29 mg/L.The results of stability showed that plasma specimens for dTT could not be stored at room temperature more than 4 hours,at 4 ℃ more than 4 days,at-20 ℃ exceed 1 month,while at-80℃ the plasma specimens could be stored at least 6 months for dTT assay.Conclusion The precision,trueness,analytical measurement range,carryover rate,anti-biological interference of dTT assay may meet the requirement of clinical laboratory.The stability of the specimen can fulfill the clinical requirements.

OBJECTIVETo investigate the mechanism of low glucose-induced injury in human ECV304 cells.

METHODSHuman umbilical vein endothelial cell line ECV304 were stimulated with low concentrations of glucose. The level of reactive oxygen species (ROS) in cells was detected at different time points within 12 h by kinetic measurement of dichlorofluorescein (DCF) fluorescence produced by oxidation of an oxidant-sensitive dye 2,7-dichlorofluorescein (DCFH). ECV304 cell viability was assessed with MTT assay and NADPH oxidase activity detected using lucigenin-enhanced chemiluminescence assay following cell stimulation with low glucose and apocynin.

RESULTSLow-glucose exposure of ECV304 cells time- and dose-dependently induced ROS production, and which was decreased by apocynin treatment. Apocynin pretreatment of the cells inhibited ROS production by 44% in cells exposed to 2.8 mmol/L glucose and by 60% in cells without glucose exposure.

CONCLUSIONSLow glucose of ECV304 cells induces ROS production to cause cell injury, which is mediated partially by NADPH oxidase activation.

Cell Line ; Glucose ; administration & dosage ; adverse effects ; Humans ; NADPH Oxidases ; metabolism ; Oxidative Stress ; drug effects ; Reactive Oxygen Species ; metabolism

OBJECTIVETo investigate the change of indicators of oxidative stress in serum and NF-kappaB in peripheral blood mononuclear cells of patients with silicosis, and explore the mechanism of the development of silicosis.

METHODSThe subjects were divided into (1) 200 workers exposed to SiO2 for at least 1 years in a foundry served as the dust-exposure group; (2) 130 cases with silicosis (I phase silicosis 64 cases, II phase 46 cases III phase 20 cases) served as the silicosis group; (3) 32 cases with 0+ phase silicosis in the foundry served as the observed group,(4)100 subjects from a hotel served as the control group. The serum including superoxide dismutase (SOD), nitric oxide (NO), serum glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), nitric oxide synthase (NOS), lipid malondialdehyde (MDA) and NF-kappaB protein levels in peripheral blood mononuclear cells were determined, respectively.

RESULTSCompared with the control group, NO levels in dust-exposed group and silicosis group significantly increased, and SOD decreased significantly (P < 0.05 or P < 0.01). Compared with the control group and dust-exposed group, T-AOC, NOS, MDA levels in silicosis group significantly increased (P < 0.05 or P < 0.01). GSH-Px in dust-exposed group and silicosis group were (231.164 +/- 36.484) and (270.469 +/- 39.228)U/ml, respectively which were significantly than that [(223.360 +/- 46.838) U/ml] in control group (P < 0.05 or P < 0.01), and there was significant difference of GSHPx between the silicosis group and the dust-exposed group significantly (P < 0.01) . GSH-Px level [(290.750 +/- 39.129) U/ml] in III phase silicosis group were significantly higher than those [(256.906 +/- 21.41) and (259.594 +/- 34.79) U/ml] in observation group and I phase silicosis group (P < 0.05). NF-kappaB levels [(72.06 +/- 9.12) and (85.25 +/- 11.64) ng/L] in dust-exposed group and silicosis group were significantly higher than that [(59.71 +/- 9.27) ng/L] in control group (P < 0.01), and there was significant difference of between the silicosis group and the dust-exposed group (P < 0.01). There was a positive correlation between serum GSH-Px level and the silicosis stages (r = 0.507, P < 0.01). Also there was a positive correlation between NF-kappaB level and silicosis stages, age, GSH-Px or NO levels (r = 0.376, 0.243, 0.233, 0.221, P < 0.01).

CONCLUSIONThe imbalance of oxidative and anti-oxidation system and the activation of NF-kappaB are related with the occurrence and development of silicosis. The monitoring of oxidative stress indicators and NF-kappaB is beneficial to the prediction and prognosis assessment of silicosis.

Adult ; Aged ; Case-Control Studies ; Glutathione Peroxidase ; metabolism ; Humans ; Leukocytes, Mononuclear ; metabolism ; Malondialdehyde ; metabolism ; Middle Aged ; NF-kappa B ; metabolism ; Nitric Oxide ; metabolism ; Oxidative Stress ; Silicosis ; blood ; Superoxide Dismutase ; metabolism ; Young Adult

Objective Prevalence of occult hepatitis B virus (HBV) infection (OBI) was investigated in a paired mother-teenager population and HBV S gene variation including overt and occult HBV,was determined.Methods A follow-up study based on an initial survey of 135 mother-teenager pairs was carried out through collection of questionnaires and blood samples HBsAg were detected by ELISA method,viral load by PCR amplification and HBV S gene by phylogenetic analysis.Results 102 pairs of subjects were followed-up.Blood samples from 94 mothers and 101children were collected.OBI prevalence in mothers was 10.0％ (6/60),significantly higher than 2.0％(2/101) in teenagers.Medians of viral load were 399.9 IU/ml and 247.6 IU/ml in overt and occult HBV strains,but without significant difference.1 occult HBV strain belonged to genotype B with serotype adw while the other 7 were genotype C with serotype adr.15 of the overt HBV strains belonged to genotype B with serotype adw and the other 8 were genotype C with serotype adr.Proportions of genotype-C strains were significantly higher in occult HBV strains than in overt HBV strains.Conclusion OBI was seen in teenage-mother population.

A switch thrombin aptamer sensor was constructed based on the host-guest competition mode ofβ-cyclodextrin (β-CD ) . The aptamer that modified with ferrocene ( Fc ) at its terminal was fixed on the surface of the gold electrode via the host-guest recognization with β-CD. When thrombin was present, the configuration of aptamer transformed from vertical linear to "G-quadruplex" and far away from the electrode surface, which resulted in a decrease in the redox current of the aptamer probe and produce "Signal-off"effect. On the basis of this, a high sensitive detection of thrombin was made. The detection result indicated that the thrombin concentration had a consideration linear response to the signal of aptasensor between 5 . 0 × 10-13-5. 0 × 10-9 mol/L, and as low as 2. 0 × 10-13 mol/L thrombin had been detected. This method for thrombin detection showed a higher specificity compared to other protein molecules. Besides, the sensor was constructed easily and possessed excellent regeneration, which provided a significant platform for highly efficient real-time detection of thrombin in biological serum samples.