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| 1 | Establishment of a P-glycoprotein substrate screening model and its preliminary application显示文摘AIM: To establish a high P-glycoprotein (P-gp) expressing cell line as a model for studying drug absorption and distribution, and to explore the preliminary application of this screening model.METHODS: A full-length MDR1 cDNA fragment in plasmid pMDRA1 was first subcloned into plasmid pET28a(+), then MDR1 cDNA was cut from the recombinant plasmid with double-digestion and ligated into the mammalian expression vector pcDNA3.1(+). The recombinant plasmid pcDNA3.1(+)/MDR1 was transfected into breast cancer cell line Bcap37 using the Superfect transfection reagent. Several stably transfected clones were obtained after selection with G418.Real-time fluorescent quantitative RT- PCR and Western blot methods were used to detect the expression of P-gp, and the cellular location of the expressed protein was determined by immunohistochemical staining. Drug sensitivity assay was used to evaluate the biological function of expressed P-gp.Concentration of quercetin in cells was determined by high-performance liquid chromatography (HPLC).RESULTS: The recombinant plasmid was confirmed to be inserted in the correct orientation by restrictive enzyme digestion and DNA sequencing. Real-time fluorescent quantitative RT-PCR showed a higher level of P-gp mRNA in transfected cells compared to that in the control cells, and the Western blot result also indicated that P-gp expression in transfected cells was higher than that in control cells. The immunohistochemical staining showed that the expressed P-gp was localized on cell membranes. Drug sensitivity assay showed that the ICs0 for adriamycin and colchicine of the transfected cells was higher than that of the control cells.The concentration of quercetin in model cells was lower than that in control cells by HPLC. After P-gp inhibitor verapamil was administered, the concentration of quercetin in model cells was increased.CONCLUSION: A high P-gp expressing cell line can be established, which could provide a suitable in vitro model system for studying drug intestinal absorption mechanism,predicting the drug permeability characteristics and screening new multi-drug resistance reversing agents. With this model,quercetin can be found to be transported by P-gp, and it is a P-gp substrate. | YiWang JiangCao SuZeng | 2004 | World Journal of Gastroenterology2004，10，9： | 6 |
| 2 | Determination of gemcitabine and its metabolite in human plasma using high-pressure liquid chromatography coupled with a diode array detector显示文摘AIM: To establish a high pressure liquid chromatography (HPLC) method for determination of the concentration of gemcitabine (dFdC) and its metabolite(dFdU) in human plasma. METHODS: Plasma 1.0 mL spikedwith floxuridine as an internal standard was extracted with 3.0 mL of methanol-acetonitrile (v/v, 1:9). The supernatant was evapo- rated at 60 oC and the residue was reconstituted with 0.5 mL of the solution used as the mobile phase. After centrifugation, 50 μL of the supernatant was injected into the HPLC system. Separation was achieved on a C18 (4.6 mm×250 mm, 5 μm) column at 25 oC with the flow rate of the mobile phase set to 0.8 mL/min. The compounds were detected at 268 nm. The mobile phase consisted of 40.0 mmol/L acetate ammonium buffer solution (pH 5.5) and acetonitrile (v/v, 97.5:2.5). RESULTS: The linear range was 0.20-10.0 mg/L(r=0.9999) for dFdC and 0.50-50.0 mg/L(r=0.9999) for dFdU. The limit of detection (LOD) was 0.10 mg/L for dFdC and 0.25 mg/L for dFdU, while the limit of quantification (LOQ) was 0.20 mg/L (RSD<10 %) for dFdC and 0.50 mg/L(RSD <3 %) for dFdU. The average recovery of dFdC and dFdU by this method were 103.3 % and 98.7 %, respectively. For intra-day and inter-day, the corresponding standard deviations of the measurements of dFdC and dFdU were both less than 5.5 %. CONCLUSION: An analytical method was established to measure the concentrations of dFdC and dFdU in human plasma and was effectively applied to the dFdC and dFdU pharmacokinetic studies of 8 Chinese patients with malignant tumors. | Neng-mingLIN SuZENG Sheng-linMA YunFAN Hai-junZHONG LuoFANG | 2004 | Acta Pharmacologica Sinica2004，25，12： | 4 |
| 3 | Heterologous expression of active human undine diphosphate glucuronosyltransferase 1A3 in Chinese hamster lung cells显示文摘AIM: To obtain the active human recombinant uridine diphosphate glucuronosyltransferase 1A3 (UGT1A3) enzyme from Chinese hamster lung (CHL) cells.METHODS: The full-length UGT1A3 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR)using total RNA from human liver as template. The correct fragment confirmed by sequencing was subcloned into the mammalian expression vector pcDNA3.1 (+), and the recombinant vector was transfected into CHL cells using a calcium phosphate method. Expressed UGT1A3 protein was prepared from CHL cells resistant to neomycin (G418). Then the protein was added into a reaction mixture for glucuronidation of quercetin. The glucuronidation activity of UGT1A3 was determined by reverse phase-high performance liquid chromatography (RP-HPLC) coupled with a diode array detector (DAD). The quercetin glucuronide was confirmed by hydrolysis with β-glucuronidase. Control experiments were performed in parallel. The transcriptions of recombinants were also determined by RT-PCR.RESULTS: The gene was confirmed to be an allele (UGT1A3-3) of UGT1A3 by DNA sequencing. The fragment was introduced into pcDNA3.1 (+) successfully. Several colonies were obtained under the selection pressure of G418.The result of RT-PCR showed transcription of recombinants in mRNA level. Glucuronidation assay and HPLC analysis indicated UGT1A3 expressed heterologously in CHL cells was in an active form, and one of the gulcuronides corresponding to quercetin was also detected.CONCLUSION: Correct sequence of UGT1A3 gene can be obtained, and active UGT1A3 enzyme is expressed heterologously in CHL cells. | Ya-KunChen XinLi Shu-QingChen SuZeng | 2005 | World Journal of Gastroenterology2005，11，1： | 3 |
| 4 | Nuclear Localization of Annexin A1 Correlates With Advanced Disease and Peritoneal Dissemination in Patients with Gastric Carcinoma显示文摘 | FengjiaZhu ChaoyangXu ZhinongJiang MeiJin LinboWang SuZeng LisongTeng JiangCao | 2010 | Anat Rec2010，，8： | 1 |
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