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徐顺清, Xi Sun, Fang Li, You-jie Wang, Yi-rong Li, Yan-hua Su, Yuan-yuan Li, Hong Yan, and Shun-qing Xu
TOXICOLOGICAL SCIENCES 80, 49-53 (2004),-0001,():
-1年11月30日
The aromatic hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biological and toxicological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related chemicals. Here we developed a novel method to detect the presence of AhR ligands using Exonuclease Protection Mediated PCR bioassay (EPM-PCR). This assay measures the ability of a chemical to activate AhR DNA binding in vitro. In the presence of AhR ligand, an expected length PCR product was observed on electrophoresis, but no signal was detected in the absence of ligand. Real-time quantitative PCR was performed to quantifyDNA bound to ligand:AhR complex. We obtained a standard curve with TCDD concentration to bound DNA copies in the range of 0.01pM-10nM of TCDD. Minimal detection limit of the assay was below 0.01pM TCDD, and the whole detection time was less than 5h. In comparison to the chemical-activated luciferase gene expression (CALUX) bioassay, EPM-PCR bioassay is more sensitive and easier to perform. These results suggest that this assay is useful for detection and quantification of TCDD and related AhR ligands in a cell-free system without the use of radioactivity.
Ah receptor, 2,, 3,, 7,, 8- TCDD, exonuclease Ⅲ, S1 nuclase, real-time PCR.,
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【期刊论文】Cyclooxygenase-2 expression in squamous dysplasia and squamous cell carcinoma of the esophagus
徐顺清, Hong-Ping Yu a, Shun-Qing Xu a, *, Li Liu b, Lu-Yuan Shi c, Xiao-Kun Cai a, Wen-Hong Lu a, Bin Lu a, Yan-Hua Su a, Yuan-Yuan Li a
Cancer Letters 198(2003)193-201,-0001,():
-1年11月30日
Cyclooxygenase-2 (cox-2) overexpression has been observed in several types of human cancers and has been implicated in carcinogenesis. To elucidate the role of cox-2 in esophageal carcinogenesis, we evaluated the expression of cox-2 in normal squamous epithelium squamous epithelial dysplasia
Cyclooxygenase-2, Esophagus, Squamous epithelial dysplasia, Squamous cell carcinoma, Immunohistochemistry
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徐顺清, Zhi-Ren Zhang, Shun-Qing Xu, Xi Sun, Yong-Jun Xu, Xiao-Kun Cai, Zhi-Wei Liu, Xiang-Lin Tan, Yi-Kai Zhou, Jun-Yue Zhang, Hong Yan, Zhi-Ren Zhang
World J Gastroenterol 2003; 9 (7): 1460-1464,-0001,():
-1年11月30日
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 pGL3-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 HepG2, 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 doseeffect relationships of inducible luciferase expression to DLCs was dynamically observed in HepG2 cells stably transfected by the recombinant vector (HepG2-Luc) and compared with that assayed by ethoxyresorufin-O-deethylase (EROD) in non-transfected HepG2 cells (HepG2-wt). RESULTS: The inducible luciferase expression of HepG2-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 HepG2-Luc cells to TCDD induction was decreased with culture time and became undetectable at 10th month of HepG2-Luc cell formation. The fact that luciferase activity induced by 3, 3', 4, 4'-PCB in HepG2-Luc cells was much less than that induced by TCDD suggests a structureinducibility relationship existing among DLCs. Within the concentrations from 3.5
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【期刊论文】Bioluminescent Method for Detecting Telomerase Activity
徐顺清, Shun-Qing Xu, * Min He, Hong-Ping Yu, Xiao-Yang Wang, Xiang-Lin Tan, Bin Lu, , Xi Sun, Yi-Kai Zhou, Qun-Feng Yao, Yong-Jun Xu, and Zhi-Ren Zhang
Clinical Chemistry 48: 7 1016-1020 (2002),-0001,():
-1年11月30日
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 PPi for each TTAGGG repeat (1 pmol PPi/310 pg telomeric repeats). We developed a novel method to measure telomerase activity by use of an enzymatic luminometric PPi assay (ELIPA). Methods: Extracts of cell lines and tissues were incubated with primer at 30℃ for 30min. Released PPi 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 <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.
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徐顺清, Shunqing Xu, Min He, Hongping Yu, Xiaokun Cai, Xianglin Tan, Bin Lu, and Baihua Shu
Analytical Biochemistry 299, 188-193 (2001),-0001,():
-1年11月30日
Telomerase is expected to be a new biomarker for cancer diagnosis. The telomeric repeat amplification protocol (TRAP) is a sensitive method to detect telomerase activity. However, TRAP and its modified protocols are not always suitable for measuring telomerase activity of a large number of clinical samples to diagnosis cancer because these methods generally require a time-consuming detection step such as gel electrophoresis. To improve the procedure for mass diagnosis, we applied bioluminescence to replace the detection step. Telomerase activity is measured by evaluating the amount of inorganic pyrophosphate generated in PCR amplification of telomerase elongation product, with use of the sensitive enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA). TRAP connected with ELIDA (TRAP-ELIDA) can quantitatively detect telomerase activity within linearity from 2 to 1000 cell equivalents. The ELIDA signals accorded with results of TRAP-SYBR green staining, and the results of ELIDA were significantly correlated to those of TRAP connected with an enzymelinked immunosorbent assay (TRAP-ELISA) (250.992, P<0.001). TRAP-ELIDA is a simple and sensitive method to quantify telomerase activity without time-consuming gel electrophoresis. Because TRAP-ELIDA measures telomerase activity with a luminometer, it could be applied to a large number of clinical samples at the same time.
telomerase, TRAP assay, bioluminescence, pyrophosphate.,
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