Objective To establish a network laboratory for blood cell analysis and better calibrate haematology analyzers in local lab.Methods According to GB/T 15481《General requirements for the competence testing and calibration laboratories》(idt ISO/IEC 17025),we established a network laboratory providing traceability for blood cell analysis.Complete blood count was traced to Calibration Laboratory in NCCL;The secondary standard haematology analyzer with the same model and calibrator with same lot number were used for verification for a long period.Fresh blood from healthy people was used to calibrate haematology analyzers.Results Gradually we have improved our laboratory quality management system, precision as well as accuracy,which was satisfactory.The unified blood sample was adopted to calibrate different equipments in our hospital and showed consistence when compared with calibration analyzer.The correlation coefficient of all tests is more than 0.99.The relative deviation of WBC,RBC,HCT,HGB and PLT are within?7%,?3.5%,?4%,?3% and?15%,respectively.Conclusions Secondary standard systems provides good comparable results with calibration laboratory.Its tracing mode and quality control scheme could ensure the traceability and accuracy of completed blood count.Furthermore,using elective fresh blood from healthy people,the comparable results from different analyzers were achievable.

To figure out the stability and intestinal bacteria metabolites of rats in vitro of astragaloside IV ( AST), this research was done to explore the stability of AST in the artificial gastric juice. artificial intestinal juice and rat liver homogenate and the metabolism in rat intestinal in vitro. HPLC was used to calculate the remaining rate of AST in biological samples by measuring the content of AST, while metabolites were determined by combining the methods of TLC, HPLC and LC-MS/MS. It turned out that AST was difficult to metabolize in the artificial gastric juice, artificial intestinal juice and rat liver. Also, the metabolic pathway of AST was stepped by deglycosylation. Firstly, AST was converted to its secondary etabolites (6-O-β-D-glucopyranosyl- cycloastragenol, CMG) by removal of xylose moiety at C-3, then transformed into cycloastragenol (CAG) after hydrolytic removal of the glucose moiety at C-6. All the results suggested that the metabolism of AST in vivo occurs mainly in the intestinal by hydrolysis of glycosyl. In conclusion, hydrolysis of intestinal flora is the main reason that AST metabolizes.
Animals ; Bacteria ; metabolism ; Chromatography, High Pressure Liquid ; Drug Stability ; Intestines ; microbiology ; Liver ; metabolism ; Rats ; Rats, Sprague-Dawley ; Saponins ; chemistry ; metabolism ; Tandem Mass Spectrometry ; Triterpenes ; chemistry ; metabolism

Epiberberine, one of the most important isoquinoline alkaloid in Coptidis Rhizoma, possesses extensive pharmacological activities. In this paper, the liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to study phase I and phase II metabolites. A Thermo HPLC system (including Surveyor AS, Surveyor LC Pump, Surveyor PDA. USA) was used. The cocktail probe drugs method was imposed to determine the content change of metoprolol, dapsone, phenacetin, chlorzoxazone and tolbutamide simultaneously for evaluating the activity of CYP2D6, CYP3A4, CYP1A2, CYP2E1 and CYP2C9 under different concentrations of epiberberine in rat liver microsomes. The result showed that epiberberine may have phase I and phase II metabolism in the rat liver and two metabolites in phase I and three metabolites in phase II are identified in the temperature incubation system of in vitro liver microsomes. Epiberberine showed significant inhibition on CYP2D6 with IC50 value of 35.22 μmol L(-1), but had no obvious inhibiting effect on the activities of CYP3A4, CYP1A2, CYP2E1 and CYP2C9. The results indicated that epiberberine may be caused drug interactions based on CYP2D6 enzyme. This study aims to provide a reliable experimental basis for its further research and development of epiberberine.
Animals ; Berberine ; analogs & derivatives ; chemistry ; metabolism ; Chromatography, High Pressure Liquid ; Cytochrome P-450 CYP2D6 ; metabolism ; Cytochrome P-450 CYP2D6 Inhibitors ; chemistry ; metabolism ; Drugs, Chinese Herbal ; chemistry ; metabolism ; Male ; Microsomes, Liver ; drug effects ; enzymology ; metabolism ; Molecular Structure ; Rats ; Rats, Sprague-Dawley ; Tandem Mass Spectrometry

OBJECTIVETo detect the loss of heterozygosity (LOH) in esophageal squamous cell carcinoma and adjacent high-grade squamous dysplasia, and to evaluate possible tumor suppressor genes in the development and progression of invasive malignancy.

METHODSLOH was detected in normal esophageal mucosa, high grade squamous dysplasia and esophageal squamous cell carcinoma using microdissection and polymerase chain reaction technology. The changes of LOH at seven microsatellite markers and the relationship between LOH rate and clinicopathologic parameters were analyzed.

RESULTSIn high grade squamous dysplasia, LOH was detected at D13S802 (40%), D13S267 (32%), D13S221 (31%), D9S942 (30%), D17S520 (24%) and D9S171 (33%). However, D17S1798 LOH was not detected. In invasive squamous cell carcinoma, LOH was detected as follows: D13S267 (71%), D13S802 (58%), D17S520 (55%), D13S221 (45%), D9S942 (43%), D9S171 (33%) and D17S1798 (11%). The frequency of LOH in the seven microsatellite markers, the pathologic grade, clinical stage and occurrence of lymph node metastasis did not show any statistically significant correlation (P > 0.05).

CONCLUSIONSThe progression from normal squamous epithelium to high grade squamous dysplasia and subsequently to invasive squamous cell carcinoma of the esophagus was associated with accumulation of genetic errors. Possible tumor suppressor genes related to the development of esophageal squamous cell carcinoma may exist near D13S802 (13q12.12). Possible tumor suppressor genes near D13S267 (13q13.1), D17S1798 (17p13.3) and D17S520 (17p13.1) may be related to the progression of esophageal squamous cell carcinoma.

Adult ; Aged ; Carcinoma, Squamous Cell ; genetics ; Chromosome Mapping ; Chromosomes, Human, Pair 13 ; Chromosomes, Human, Pair 17 ; Chromosomes, Human, Pair 9 ; Esophageal Neoplasms ; genetics ; Female ; Genes, Retinoblastoma ; Genes, Tumor Suppressor ; Genes, p16 ; Genes, p53 ; Humans ; Loss of Heterozygosity ; Male ; Microsatellite Repeats ; Middle Aged ; Precancerous Conditions ; genetics

BACKGROUNDOxidative stress plays an important role in the pathogenesis of epidermal diseases. This study aimed to investigate the effects of quercetin on the anti-oxidative response and on mitochondrial protection in cultured normal human keratinocytes.

METHODSCultured HaCaT cells were treated with different concentrations of H2O2 (0, 50, 100, 250, 500 micromol/L) for different periods of time (0.5, 1, 2, 4 hours) to establish an oxidative stress model. The cultured HaCaT cells were randomly assigned to control, H2O2, and quercetin + H2O2 groups. For the quercetin groups, the cells were treated with different concentrations of quercetin (0, 10, 25, 50 micromol/L) before exposure to H2O2. Morphological changes of the cells were observed under an inverted microscope and an electron microscope. The cell viability was detected by the MTT method. The cell apoptosis (AnnexinV/propidium iodide double stain) and mitochondrial membrane potential (DeltaPsim) changes were detected by flow cytometry.

RESULTSAn oxidative stress model of HaCaT cells was established under a suitable concentration (250 micromol/L) and treated time of H2O2 (2 hours). The cell viability and DeltaPsim decreased in a concentration-dependent and time-dependent manner while the percentage of apoptotic cells significantly increased in the H2O2 groups compared with the control group (P < 0.05). The cell viability and DeltaPsim of the quercetin treated group increased (P < 0.05) and the percentage of apoptotic cells decreased at concentrations of 1-50 micromol/L quercetin (P < 0.01) compared with H2O2 treated group.

CONCLUSIONQuercetin can relieve the cell damage and apoptosis from H2O2 induced injury to HaCaT cells by anti-oxidation and mitochondrial protection.

Antioxidants ; pharmacology ; Apoptosis ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Humans ; Hydrogen Peroxide ; toxicity ; Keratinocytes ; drug effects ; pathology ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; Quercetin ; pharmacology

A quantitative real-time PCR assay was developed to measure the proviral load of human T-lymphotropic virus type I (HTLV-I) in peripheral blood. The technology utilizes special primers and Taqman MGB fluorescence probe to measure amplification products from the gag-pro-pol polyprotein gene of HTLV-I. HTLV-I copy number was normalized to the amount of cellular DNA by quantitation of the beta-actin gene, The amplification system was sensitive to detect 5 copy/microL. The standard curve had a good linearity when the quantity for the gene was between 10(3) and 10(7) copy/microL (R2 = 0.999). Good reproducibility was observed in each intra- and inter-assay. We also measured proviral load in peripheral blood in 12 HTLV-I seropositive former blood donors. Proviral load for HTLV-I infected donors ranged from 0.015 to 12.819 copy/cell in WBC with the mean of 3.116 copy/cell.
Gene Products, gag ; genetics ; Gene Products, pol ; genetics ; Human T-lymphotropic virus 1 ; genetics ; isolation & purification ; Humans ; Molecular Probes ; Polymerase Chain Reaction ; methods ; Viral Proteins ; genetics

OBJECTIVETo evaluate the influence of VPA treatment on neutrophils' oxidative metabolism and oxidant status in epileptic children.

METHODTwenty-six newly diagnosed epileptic children with idiopathic epilepsy and 30 healthy children were included in the study. The activation rates of neutrophils and stimulation indexes were detected in patients before and 6 months and 12 months after VPA treatment respectively and in all the healthy children by flow cytometry with dihydrorhodamine as fluorochrome. The activities of myeloperoxidase from neutrophils were also detected. Malondialdehyde as an indicator of lipid peroxidation and antioxidant enzymes including superoxide dismutase, catalase, and glutathione peroxidase were measured in plasma respectively.

RESULTThe activation rates of neutrophils in patients treated with VPA after 6 and 12 months were (11.50 ± 6.52)% and (14.31 ± 5.76)% respectively, which were significantly higher than the data of control group (5.90 ± 3.77)% and pretreatment level (7.42 ± 3.15)%. The stimulation indexes 6 and 12 months after VPA therapy were (474.88 ± 118.98) and (416.31 ± 110.00) respectively, which were lower than the data of control group (544.83 ± 140.83) and pretreatment level (535.23 ± 111.55). The plasma MPO activities and levels of malondialdehyde in VPA treated patients were also higher while the activities of SOD and CAT were significantly lower than the control and untreated groups. GSH-Px levels did not differ between the groups. Multiple linear regression analysis showed that the time of treatment and the activation rates of neutrophils were indicators which had positive correlation with the levels of plasma MDA and that SOD activities were inversely correlated with MDA levels.

CONCLUSIONVPA which is frequently used in childhood epilepsy may activate the neutrophils of patients and cause oxidative stress and prolonged treatment may aggravate it.

Anticonvulsants ; pharmacology ; therapeutic use ; Antioxidants ; pharmacology ; Case-Control Studies ; Catalase ; blood ; Child ; Child, Preschool ; Epilepsy ; blood ; drug therapy ; Female ; Glutathione Peroxidase ; blood ; Humans ; Lipid Peroxidation ; drug effects ; Male ; Malondialdehyde ; blood ; Neutrophils ; drug effects ; metabolism ; Oxidative Stress ; drug effects ; Superoxide Dismutase ; blood ; Valproic Acid ; pharmacology ; therapeutic use

OBJECTIVETo find a convenient and exact method for evaluating acrosome reaction in human spermatozoa.

METHODSThe semen of the normal male was mixed and then divided into 6 groups. Coomassie brilliant blue (CBB) staining, chlortetracycline (CTC) fluorescence staining and acid phosphatase (ACP) detection were used for morphological observation and data analysis of the acrosome status of the human sperm treated with or without progesterone.

RESULTSThere were obvious morphological differences between the acrosome-reaction and acrosome-intact spermatozoa in CBB staining and CTC fluorescence staining, and significant differences were observed between the experimental and control spermatozoa by the three methods (P < 0.05).

CONCLUSIONAll the three methods can be used to assess acrosome reaction in human spermatozoa, but Coomassie brilliant blue (CBB) staining is much more convenient and stable.

Acid Phosphatase ; Acrosome Reaction ; drug effects ; Cells, Cultured ; Chlortetracycline ; Humans ; Male ; Progesterone ; pharmacology ; Rosaniline Dyes ; Spermatozoa ; cytology ; Staining and Labeling ; methods

OBJECTIVETo investigate the relationship of leptin gene polymorphism with obesity in ethnic minority Hui and Uygur children in China.

METHODSSixty-eight ethnic minority (35 Hui and 33 Uygur) children with obesity and 69 age-matched minority (36 Hui and 33 Uygur) children without obesity were recruited from six primary schools in the sub-urban areas of Urumqi. Venous blood was sampled from all subjects after fasting for 12 hours. Leptin gene C2549A polymorphism was determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism methods. Blood concentrations of lipids, leptin and insulin were measured with biochemical methods and radioimmunoassys, respectively.

RESULTSIn the 137 children tested, the prevalence of AA, AC and CC genotype was 9.5%, 33.6% and 56.9%, respectively. A allele frequency was significantly different between the two ethnic (i.e. Hui and Uygur) groups (P<0.05). A allele frequency and AA+ AC genotype frequency were not significantly different between obese and non-obese children in both ethnic groups (P>0.05). Blood leptin levels were not significantly different between obese and non-obese children with an AA+AC or CC genotype in both ethnic groups (P>0.05).

CONCLUSIONSLeptin gene polymorphisms exist in Hui and Uygur children. The C2549A polymorphism is not significantly associated with the prevalence of obesity in both Hui and Uygur children.

Child ; China ; ethnology ; Female ; Genotype ; Humans ; Leptin ; blood ; genetics ; Lipids ; blood ; Male ; Obesity ; blood ; genetics ; Polymorphism, Genetic

OBJECTIVES: Some changes are found in the labyrinth anatomy during postnatal development. Although the spatial orientation of semicircular canals was thought to be stable after birth, we investigated the age-related orientational changes of human semicircular canals during development. METHODS: We retrospectively studied the computed tomography (CT) images of both ears of 76 subjects ranged from 1 to 70 years old. They were divided into 4 groups: group A (1-6 years), group B (7-12 years), group C (13-18 years), and group D (>18 years). The anatomical landmarks of the inner ear structures were determined from CT images. Their coordinates were imported into MATLAB software for calculating the semicircular canals orientation, angles between semicircular canal planes and the jugular bulb (JB) position. Differences between age groups were analyzed using multivariate statistics. Relationships between variables were analyzed using Pearson analysis. RESULTS: The angle between the anterior semicircular canal plane and the coronal plane, and the angle between the horizontal semicircular canal plane and the coronal plane were smaller in group D than those in group A (P<0.05). The JB position, especially the anteroposterior position of right JB, correlated to the semicircular canals orientation (P<0.05). However, no statistically significant differences in the angles between ipsilateral canal planes among different age groups were found. CONCLUSION: The semicircular canals had tendencies to tilt anteriorly simultaneously as a whole with age. The JB position correlated to the spatial arrangement of semicircular canals, especially the right JB. Our calculation method helps detect developmental and pathological changes in vestibular anatomy.
Ear ; Ear, Inner ; Humans* ; Methods ; Parturition ; Retrospective Studies ; Semicircular Canals* ; Tomography, X-Ray Computed