Biomedical subjects

Yi-Kai Zhou

Publications and source records attributed to Yi-Kai Zhou.

10 recordsLinked to original sources

PubMed2006

Selective solid-phase extraction of cholesterol using molecularly imprinted polymers and its application in different biological samples.

Non-covalent molecularly imprinted polymers (MIPs) of cholesterol were prepared by UV initiated polymerization. A polymer that had the highest binding selectivity and capability was used as solid-phase extraction (SPE) sorbents for direct extraction of cholesterol from different biological samples (human serum, cow milk, yolk, shrimp, pork and beef). The extraction conditions of molecularly imprinted SPE (MISPE) were optimized and the optimum protocol was: conditioning MISPE cartridges with n-hexane, loading with n-hexane, washing with n-hexane and n-hexane:toluene=9:1, respectively, then eluting with chloroform:ethanol:acetic acid=3:1:1. Cholesterol MISPE selectively recognized, effectively trapped and pre-concentrated cholesterol over a concentration range of 10-80 microg/mL. Recoveries ranged from 80.6% to 92.7%, with R.S.D. lower than 9.8%. Under the optimal condition, MISPE recoveries of spiked human serum, yolk, cow milk, shrimp, pork and beef were 91.1%, 80.4%, 86.6%, 78.2%, 81.4% and 80.1%, respectively. Compared with C18 SPE, almost all of the matrix interferences were removed after MISPE, and better baselines and higher selectivity were achieved.

Animals↗

PubMed2006

In vitro cytotoxicity of TCDD on SPC-A1 cells.

OBJECTIVE: The toxicology of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) has been studied mainly with regard to the carcinogenicity of its metabolites, but its phototoxicity is not well understood. Although some studies have indicated the lethal phototoxicity of TCDD, this study was designed to investigate its effect on SPC-A1 cells. METHODS: SPC-A1 cells were cultured in 1640 medium and treated with 10 nmol/L, 0.1 micromol/L, 1 micromol/L TCDD for either 24 h or 96 h at each concentration. SPC-A1 cells were co-cultured with TCDD at different concentrations. Then the cell morphology, DNA fragment electrophoresis, and cell cycle were analyzed by flow cytometry, and enzyme assays were used to observe the effect of TCDD on the morphology, growth rate, and enxyme change of SPC-A1 cells. RESULTS: With the increasing concentrations of TCDD and prolongation of culture time, the morphology of SPC-A1 cells was changed from round shape to spindle, and the ability of SPC-A1 cells to adhere to wall was decreased. With debris emitted around the cells, the morphologic changes included reduction in cell volume. Nuclear chromatin condensation and PI were observed. With the increasing concentrations of TCDD, DNA ladder occurred. After treatment with TCDD, extraction of cancer cells exhibited typical DNA fragmentation, and flow cytometry analysis showed apoptosis in a dose-dependent manner. As the concentration of TCDD rose from 10 nmol/L to 1 micromol/L, the ratio of apoptotic cells increased from 10.76% to 21.82%. CONCLUSIONS: TCDD has in vitro cytotoxicity on SPC-A1 cells, and the cytotoxicity is positively related to its concentration and culture time. TCDD may inhibit the growth and proliferation of SPC-A1 cells through the pathway of apoptosis introduction.

Cell Line, Tumor↗

PubMed2005

A quantitative DNA methylation assay using mismatch hybridization and chemiluminescence.

OBJECTIVE: To develop a quantitative method for methylation analysis of the p16 gene based on mismatch hybridization and chemiluminescence. METHODS: Genomic DNA was modified by sodium bisulfite to convert all unmethylated but not methylated cytosines to uracil, and subsequently a pair of primer having no CpG sites was designed for amplification target DNA containing methylated or unmethylated CpG sites. The PCR product spanning CpG sites were hybridized with two oligonucleotide probes which perfectly matched the methylated and unmethylated CpG sequences respectively, and the hybrids were detected by chemiluminescent method. The percentage of methylated target sequences could be estimated by calculating the ratio of signals obtained with two probes. RESULTS: The percentage of methylation of artificial mixtures DNA showed a linear relation. There was a negative correlation between the methyaltion index with p16 transcriptional mRNA of p16 gene in tumor cell lines. CONCLUSION: Compared with existing methods, this assay is nonisotopic, rapid, simple, and can be widely applied to the study of DNA methylation.

Cell Line, Tumor↗

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↗

PubMed2003

[Construction of nucleotide excision repair gene XPB antisense RNA expression plasmid and its functions].

BACKGROUND & OBJECTIVE: Nucleotide excision repair is an important pathway for cellular DNA damage repair. The drug resistance of tumor cell is often companied with the enhanced expression of DNA repair genes. Down-regulation of DNA repair capacity by antisense strategy can increase the drug sensitivity of tumor cells. The aim of this study was to construct the eukaryotic expression plasmid pcDNA-XPB/AS (XPB: xeroderma pigmentosum B) and to investigate the function of XPB gene and its roles in chemotherapeutic drug sensitivity in lung cancer A549 cell. METHODS: The XPB cDNA (69-520 bp) fragment amplified by reverse transcription polymerase chain reaction (RT-PCR) was inserted into pcDNA3.1/His plasmid with an inverted orientation. The recombinant plasmid was transiently transfected into A549 cells. The Adriamycin-induced DNA damage was compared between the transfected and the untransfected cells by single cell gel electrophoresis assay (SCGE). The cellular sensitivity to Adriamycin of the transfected and the untransfected cells was determined by MTT assay. RESULTS: The successful construction of antisense plasmid was proved by restriction map and sequence analysis. RT-PCR results showed that the XPB mRNA expression was inhibited in transfected A549 cells. SCGE showed that the cellular damage repair ability induced by 4.0 microg/ml Adriamycin was suppressed in transfected cells. MTT assay showed the sensitivity of the transfected cells to Adriamycin was different from the untransfected cells but without statistical meaning. CONCLUSION: The antisense plasmid constructed by the authors can down-regulate the expression of XPB mRNA in the transfected cells and inhibit the cellular DNA damage repair ability, providing a basis to further study the gene function of XPB.

Base Sequence↗

PubMed2003

[Gene polymorphism of myeloperoxidase and genetic susceptibility to lung cancer].

BACKGROUND & OBJECTIVE: A-463 bp G/A polymorphism is located in the promoter region of myeloperoxidase (MPO) gene was found to be associated with lung cancer susceptibility; however, this association in Chinese population remained unclear. The aim of this study was designed to explore this association in Chinese population. METHODS: MPO genotypes in 98 cases of primary lung cancer and 112 persons of healthy control were detected using PCR-restriction fragment length polymorphism assay(PCR-RFLP) in a case-control molecular epidemiology study. The association between this gene polymorphism and the risk of lung cancer in Chinese population was examined through comparing odds ratio(OR) and 95% confidence interval(CI) between two groups. RESULTS: In healthy control group, the frequencies of persons carrying G/G, G/A, and A/A genotypes were 47.3%, 42.9%, and 9.8%, respectively. In cancer group, the frequencies of the persons carrying above three genotypes were 63.3%, 33.7%, and 3.0%, respectively. There was no significant difference of the frequencies of heterozygote G/A between two groups (P >0.05). However, The A/A genotype was found significantly higher in cancer group than that in healthy control group(P< 0.025). The risk of lung cancer for person carrying at least one A allele was only 52% of the persons carrying G/G genotype (95% CI 0.29-0.93). In smoking population, allele A showed to be a significant protective factor (OR=0.41,P< 0.025), while such protective role was not observed in non-smoking group(P >0.25). CONCLUSION: MPO gene polymorphism was associated with susceptibility of lung cancer in Chinese population. The risk of lung cancer was decreased in the persons carrying allele A.

Adult↗

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

[Catalysis of lyase-isomerase PecE/PecF for several apophycobiliproteins].

Phycoerythrocyanin(PEC) lyase-isomerase PecE/PecF from Mastigocladus laminosus is the specific enzyme for biosynthesis of PEC alpha-subunit(alpha-PEC). In this work, the specificity of PecE/PecF on substrate apoproteins was reported. PecE/PecF could catalyse the reconstitution of phycocyanobilin(PCB) with apoproteins of alpha-PEC from two different subspecies of Mastigocladus laminosus, as well the site-directed mutated apoprotein of alpha-PEC with Trp at 128 to Phe in vitro, but could not catalyse the reconstitution of PCB with apoprotein of phycocyanin alpha-subunit(alpha-CPC) from Mastigocladus laminosus. The surfactant Triton X-100 had no effect for the reconstitution of alpha-PEC, while it could improve the reconstitution of PCB with apoprotein of alpha-CPC.

Apoproteins↗