Biomedical subjects

Yong-Jun Xu

Publications and source records attributed to Yong-Jun Xu.

5 recordsLinked to original sources

PubMed2005

[Study on detection method of 8-OH-dG in DNA extracted from HepG2 cells in vitro by capillary zone electrophoresis].

OBJECTIVE: To establish an optimized method to detect 8-OH-dG after DNA oxidation damage by capillary zone electrophoresis. METHODS: HepG2 cell was used as target cell and conditions for the separation and detection were obtained by studying the influence of pH of the running buffer, temperature, running voltage on the separation. 0 and 15 mmol/L H2O2 were added into two groups of HepG2 cells (5 x 10(7)) respectively for 24h. DNA was extracted by saturated salting out method to avoid the formation of additional 8-OH-dG by the method of phenol/chloroform extraction. DNA samples were digested to free nucleotides by incubation overnight at 37 degrees C with a mixture of DNase I, snake venom phosphoatase and alkaline phosphate. Proteins were removed and the supernatant was neutralized and then extracted with diethyl ether. The residue was evaporated to dryness and reconstituted and then analyzed under the optimized conditions by capillary zone electrophoresis. RESULTS: The optimized conditions were: uncoated silica capillary (47 cm x 50 microm i.d.), 20 mmol/L borate buffer (pH 9.5), 25 degrees C, 25kV. The sample was injected by hydrostatic method for 20s. In either of H2O2-treated group and H2O2-untreated group, the peak of 8-OH-dG was detected. The 8-OH-dG content of H2O2-untreated DNA increased. CONCLUSION: The method is convenient, rapid, sensitive and cheap and safe. It provides an experimental platform to the application of 8-OH-dG in the biological monitoring of population.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2003

Characteristics and application of established luciferase hepatoma cell line that responds to dioxin-like chemicals.

AIM: To establish a luciferase reporter cell line that responds dioxin-like chemicals (DLCs) and on this basis to evaluate its characteristics and application in the determination of DLCs. METHODS: A recombinant luciferase reporter plasmid was constructed by inserting dioxin-responsive element (DREs) and MMTV promoter segments into the pGL(3)-promoter plasmid immediately upstream of the luciferase gene, which was structurally demonstrated by fragment mapping analysis in gel electrophoresis and transfected into the human hepatoma cell line HepG(2), both transiently and stably, to identify the inducible expression of luciferase by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). The time course, responsive period, sensitivity, structure-inducibility and dose-effect relationships of inducible luciferase expression to DLCs was dynamically observed in HepG(2) cells stably transfected by the recombinant vector (HepG(2)-Luc) and compared with that assayed by ethoxyresorufin-O-deethylase (EROD) in non-transfected HepG(2) cells (HepG(2)-wt). RESULTS: The inducible luciferase expression of HepG(2)-Luc cells was noted in a time-, dose-, and AhR-dependent manner, which peaked at 4 h and then decreased to a stable level at 14 h after TCDD treatment. The responsiveness of HepG(2)-Luc cells to TCDD induction was decreased with culture time and became undetectable at 10th month of HepG(2)-Luc cell formation. The fact that luciferase activity induced by 3, 3', 4, 4'-PCB in HepG(2)-Luc cells was much less than that induced by TCDD suggests a structure-inducibility relationship existing among DLCs. Within the concentrations from 3.5 x 10(-12) to 5 x 10(-9) mol/L, significant correlations between TCDD doses and EROD activities were observed in both HepG(2)-luc and HepG(2)-wt cells. The correlation between TCDD doses from 1.1 x 10(-13) to 1 x 10(-8) mol/L and luciferase activities was also found to be significant in HepG(2)-luc cells (r=0.997, P<0.001), but not in their HepG(2)-wt counterparts. For the comparison of the enzyme responsiveness between cell lines to TCDD, the luciferase sensitivity and reproducibility in HepG(2)-luc cells were both better than that of EROD in HepG(2)-wt cells, the former was at 1.1 x 10(-13) mol/L and 3.5 x 10(-12) mol/L, and the coefficients of variation (CV) of the latter was 15-30 % and 22-38 %, respectively. CONCLUSION: The luciferase expression of HepG(2)-luc cells established in the present study could sensitively respond to the DLCs stimulation and might be a prospective tool for the determination of DLCs.

Carcinoma, Hepatocellular↗

PubMed2002

Improvement of chemically-activated luciferase gene expression bioassay for detection of dioxin-like chemicals.

OBJECTIVE: To improve the chemically-activated luciferase expression (CALUX) bioassay for detection of dioxin-like chemicals (DLCs) based on the toxicity mechanisms of DLCs. METHODS: A recombinant vector was constructed and used to transfect human hepatoma (HepG2). The expression of this vector was 10-100 folds higher than that of pGL2 used in previous experiments. The transfected cells showed aromatic hydrocarbon receptor (AhR)-meditated luciferase gene expression. The reliability of luciferase induction in this cell line as a reporter of AhR-mediated toxicity was evaluated, the optimal detection time was examined and a comparison was made by using the commonly used ethoxyresoufin-O-deethylase (EROD) activity induction assay. RESULTS: The results suggested that the luciferase activity in recombinant cells was peaked at about 4 h and then decreased to a stable activity by 14 h after TCDD treatment. The detection limit of this cell line was 0.11 pmol/L, or 10-fold lower than in previous studies, with a linear range from 1 to 100 pmol/L, related coefficient of 0.997, and the coefficient of variability (CV) of 15-30%. CONCLUSION: The luciferase induction is 30-fold more sensitive than EROD induction, the detection time is 68 h shorter and the detection procedure is also simpler.

Biological Assay↗

PubMed2002

Bioluminescent method for detecting telomerase activity.

BACKGROUND: Telomerase is a promising biomarker in cancer diagnosis and therapy. The elongation of telomeric repeats catalyzed by telomerase is accompanied by release of six PP(i) for each TTAGGG repeat (1 pmol PP(i)/310 pg telomeric repeats). We developed a novel method to measure telomerase activity by use of an enzymatic luminometric PP(i) assay (ELIPA). METHODS: Extracts of cell lines and tissues were incubated with primer at 30 degrees C for 30 min. Released PP(i) was converted to ATP by sulfurylase, and ATP was detected by a luciferase bioluminescence system. The ELIPA results were compared with results obtained with the conventional telomeric repeat amplification (TRAP)-ELISA in 42 lung carcinoma tissues and 27 control tissues without malignancy. RESULTS: The lower detection limits of ELIPA and TRAP-ELISA were 5 and 10 cells, respectively. The within-run imprecision (CV) of ELIPA was < or =12%. When compared with TRAP-ELISA, the correlation coefficient (r) was 0.79. When we used the cutoff value from ROC analysis to distinguish malignant and nonmalignant tissues, the sensitivity and specificity of ELIPA were 83% and 96%, respectively, whereas the sensitivity and specificity of TRAP-ELISA were 71% and 96%, respectively. CONCLUSION: ELIPA is a simple and sensitive homogeneous method to quantify telomerase activity.

Adenosine Triphosphate↗

PubMed2002

[Investigation of progesterone receptor on human sperm plasma membrane].

OBJECTIVES: To investigate the localization and positive percentage of progesterone receptor (PR) on the human sperm surface. METHODS: After in vitro capacitation, progesterone binding sites on the sperm were quantitatively analyzed by fluorescence microscopy and flow cytometry using fluorescein isothiocyanate-labeled bovine serum albumin-progesterone complex (P-BSA-FITC). RESULTS: The spermatozoa stained by P-BSA-FITC mainly showed two labeling patterns, with the green fluorescence on the whole acrosomal region or the equatorial acrosomal region only and the stainless postacrosomal and tail regions. The percentage of progesterone-binding sperm was (30.2 +/- 2.4)%. CONCLUSIONS: There is selective expression of PR on the human sperm acrosome surface.

Adult↗