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Temporal and Tight hepatitis C Virus Gene Activation in Cultured Human hepatoma Cells Mediated by a Cell-Permeable Cre Recombinase

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Acta Biochimica et Biophysica Sinica 2004年第10期36卷 687-694页

作者： Dong XIAO*, Kang XU1, Ying YUE, Zhong-Min GUO, Bing HUANG, Xin-Yan DENG, Huan TANG2, and Xi-Gu CheN* Center of Experimental Animals, Zhongshan university, Guangzhou 510080, china 1The first affiliated hospital of Sun Yat-sen university, Zhongshan university, Guangzhou 510080, china 2.he third military medical university, chongqing 400010, china Center of Experimental Animals Zhongshan University The first affiliated hospital of Sun Yat-sen university Zhongshan University The third military medical university

Conditional gene expression has greatly facilitated the examination of the functions of particular gene products. Using the Cre/lox P switching expression system, we plan to develop efficient conditional transgeneactivation of hepatitis C virus core protein (HCV-C) cDNA (nucleotide 342–914) in the transgenic mice to overcome “immune tolerance” formed during the embryonic period and “immune escape” against hepatitis virus antigen in our project. To use this system in vivo, the dormant transgenic construct, i.e., pApoE-SCS-EGFP-HCV-C, was generated using techniques of standard molecular biology. The liver- specific human apoE promoter was here used to target expression of genes of interest (EGFP and HCV-C) to murine liver. Prior to generating the transgenic mice, the availability of Cre/lox P system and construct functionality were successfully verified by a cell-free recombination system and via checking the expression of EGFP and HCV-C in the human hepatoma cells at the mRNA and protein levels. These results suggest that the Cre/lox P system could tightly control expression of EGFP and HCV-C in vitro, which laid a solid foundation to conditionally activate expression of target gene(s) in transgenic mice by Cre-mediated site-specific recombination.

关键词： hepatitis C virus Cre/lox P switching expression system cell-free and intracellular recombination systems cell-permeable Cre recombinase EGFP human hepatoma cells

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The molecular mechanisms of the analgesic action of melatonin

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沈阳药科大学学报 2008年第S1期25卷 130-131页

作者： LI Shu-hui1,2,LI Xiao-hui1(1.Institute of Materia Medica,third military medical university,chongqing 400038,china 2.Department of Clinical Biochemistry,third military medical university,chongqing 400038,china)

Objective To analyse the potential involvement of the opioid receptor gene expression in the mechanisms of the analgesic action of *** A trauma-pain model was established in Wistar rats by combining right-hind limb amputation with 50 ℃ tail-flick *** was determined by tail-flick latency to hot waster at 50 ℃.RT-PCR was used to observe the the expression of the M1OR and KOR *** Melatonin produced the antinociceptive effect in dose-dependent manner after i.p or *** *** rats,naloxone(10 μg)obviously antagonized the antinociceptive effect induced by *** expression of the M1OR gene in the rat hypothalamus and the KOR gene in the hippocampus was both significantly reduced at day 3 after injury,which was parallel to the reduction of the rat pain ***,the expression of the M1OR gene in the hippocampus and the KOR gene in the hypothalamus was not *** of trauma-pain rats with melatonin(30-120 mg·kg-1)*** induced the up-regulation of M1OR mRNA in the hypothalamus and the KOR mRNA in the hippocampus in a concentration-dependent *** The present observations suggest that Melatonin-induced antinociceptive effect may partially contribute to the up-regulation of M1OR mRNA level in the hypothalamus and the KOR mRNA level in the hippocampus.

关键词： melatonin antinociceptive mechanism receptor

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STUDIES ON The REACTIONS OF HIPPOCAMPAL FORMATION IN PAIN MODULATION AND ACUPUNCTURE ANALGESIA

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针刺研究 1989年第Z1期 45-46页

作者： Liu Zuozhou Liao Weihong Department of Physiology, The third military medical university, chongqing, china

It has been proved that the hippocampal formation is closely related tosome behaviour reactions (such as learning and memory), visceraI activities,and neuro-endocrine regulations. However, what roles the hippocampus mayplay in pain modulation and analgesic mechanism still need further *** we have done some works in this field. The blood pressure increased accompanying hippocampal EEG's slowrhythmic activity (SRA or theta rhythm) which had a higher amplitude and alonger time course. This EEG wave was specific and could be used as one ofindexes of nociceptive response. Our experiments also demonstrated that theblood pressure increase could be induced by peripheral somatic noxious stimu-

关键词： EEG STUDIES ON The REACTIONS OF HIPPOCAMPAL FORMATION IN PAIN MODULATION AND ACUPUNCTURE ANALGESIA

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hepatitis C virus infection of human hepatoma cell line 7721 in vitro

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World Journal of Gastroenterology 2001年第5期7卷 685-689页

作者： Zhi-Qiang Song~1 Fei Hao~1 Feng Min~2 Qiao-Yu Ma~2 Guo-Dong Liu~2 Department of Dermatology~1Department of Infectious Diseases~2,Southwest Hospital,third military medical university,chongqing 400038,china Department of Dermatology Southwest Hospital Third Military Medical University Chongqing 400038 China. songzq@***

AIM: To establish a cell culture system with long-term replication of hepatitis C virus in vitro. METHODS: Human hepatoma cell line 7721 was tested for its susceptibility to HCV by incubating with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various phases during the culturing periods. The presence of HCV RNA, the expression of HCV antigens in cells and/or supernatant were examined by RTPCR, in situ hybridization and immunohistochemistry respectively. RESULTS: The intracellular HCV RNA was first detected on d2 after infection and then could be intermittently detected in both cells and supernatant over a period of at least three months. The expression of HCV NS3,CP10 antigens could be observed in cells. The fresh cells could be infected by supernatant from cultured infected cells and the transmission of viral genome from HCV-infected 7721 cells to PBMCs was also observed. CONCLUSION: The hepatoma line 7721 is not only susceptible to HCV but also supports its long-term replication in vitro.

关键词： Carcinoma, hepatocellular Liver Neoplasms Antigens, Viral Cell Division hepacivirus development hepatitis C Humans In Situ Hybridization In Vitro Phenotype RNA, Viral Research Support, Non-U.S. Gov't Tumor Cells, Cultured Virus Replication

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Opioid receptor antagonists increase [Ca2+]i in rat arterial smooth muscle cells in hemorrhagic shock

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Acta Pharmacologica Sinica 2004年第3期25卷 141-146页

作者： Li KAI,Zhong-feng WANG2,Yu-liang SHI2,3,Liang-ming LIU,De-yao HU Institute of Surgery,Department of Pharmacology,third military medical university,chongqing 400042 Key Laboratory of 2 Neurobiology,Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,Shanghai 200031,china Institute of Surgery Department of Pharmacology Third Military Medical University Key Laboratory of Neurobiology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China

AIM: To examine the effects of opioid receptor antagonists and norepinephrine on intracellular free Ca2+ concentra- tion ([Ca2+]i) in mesenteric arterial (MA) smooth muscle cells (SMC) isolated from normal and hemorrhagic shocked rats in the vascular hyporesponse stage. METHODS: The rat model of hemorrhagic shock was made by withdrawing blood to decrease the artery mean blood pressure to 3.73-4.26 kPa and keeping at the level for 3 h. [Ca2+]i of vascular smooth muscle cells (VSMC) were detected by the laser scan confocal microscopy. RESULTS: In the hyporesponse VMSC of rats in hemorrhagic shock, selective δ-, κ-, and μ-opioid receptor antagonists (naltrindole, nor-binaltorphimine, and β-funaltrexamine, 100 nmol/L) as well as norepinephrine 5 μmol/L significantly increased [Ca2+]i by 47 %±13 %, 37 %±14 %, 33 %±10 %, and 54 %±17 %, respectively, although their effects were lower than those in the normal rat cells (the increased values were 148 %±54 %, 130 %±44 %, 63 %±17 % and 110 %±38 %, respectively); and the norepinephrine-induced increase in [Ca2+]i was further augmented by three δ-, κ-, and μ-opioid receptor antagonists (50 nmol/L, respectively) application (from 52 %±16 % to 99 %±29 %, 146 %±54 % and 137 %±47 %, respectively). CONCLUSION: The disorder of [Ca2+]i regulation induced by hemorrhagic shock was mediated by opioid receptor and α-adrenoceptor, which may be partly responsible for the vascular hyporesponse, and the opioid receptor antagonists improved the response of resistance arteries to vascu- lar stimulants in decompensatory stage of hemorrhagic shock.

关键词： hemorrhagic shock vascular smooth muscle opioid receptors calcium

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Individualized immunosuppression: new strategies from pharmacokinetics,pharmacodynamics and pharmacogenomics

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hepatobiliary & Pancreatic Diseases International 2005年第3期4卷 332-338页

作者： Geng Chen and Jia-Hong Dong Army Institute of hepatobiliary Surgery, Southwest Hospital, third military medical university, chongqing 400038, china Army Institute of hepatobiliary Surgery Southwest Hospital Third Military Medical University Chongqing 400038 China. jhdong@***

The ultimate goal of transplantation is the donor-specific immune tolerance, but at least in the first 15 to 20 years of this century, immunosuppressive agents are still the determinant of clinical outcome of transplant recipients. Individualizing patient's immunosuppression to optimize the balance between therapeutic efficacy and the occurrence of adverse events poses a great challenge to physicians. DATA SOURCES:The data in this article were taken mostly from MEDLINE (2000-2004), part of which were from the research of the authors. RESULTS:Individualized immunosuppression remains a problem because of the narrow therapeutic index and wide inter- and intra-patient variation of commonly-used im- munosuppressants. Recent progress in study of pharmaco-kinetics and pharmacodynamics improved the clinical outcome of transplant recipients. More importantly, the emergence of pharmacogenomics might provide a promising and complementary tool for traditional therapeutic drug monitoring (TDM). CONCLUSIONS:Individualizing organ recipient's immunosuppression to balance the therapeutic efficacy and the adverse events represents a great challenge to transplant clinicians. Pharmacogenomics shows great promise for an interesting and hopefully better future.

关键词： immunosuppression pharmacokinetics pharmacodynamics pharmacogenomics

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Construction and Activity Assay of the Activating Transcription Factor 3 Reporter Vector pATF/CRE-luc

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Acta Biochimica et Biophysica Sinica 2006年第1期38卷 58-62页

作者： Jun-Qing XU~(1,2),Jing-Lan DENG~1,You-Sheng WU~2,Han-Yan FU~2,Rui-Hua WANG~2,Jian ZHANG~2, Fan LU~(2*),and Zhong-Liang ZHAO~(2*) ~1 Department of Naclear Medicine,Xijing Hospital,Fourth military medical university,Xi'an 710032,china ~2 Department of Biochemistry and Molecular Biology,Fourth military medical university,Xi'an 710032,china Department of Biochemistry and Molecular Biology Fourth Military Medical University Xi''an 710032 China

Activating transcription factor 3 (ATF3),a member of the activating transcription factor/cAMPresponsive element binding protein (ATF/CREB) family of transcription factors,is induced by manyphysiological *** investigate the activity of ATF/CREB in cells with physiological stresses,we developeda practical reporter vector,the plasmid pATF/CRE-luc,bearing activating transcription factor/cAMP responsiveelement (ATF/CRE) binding *** plasmid was constructed by inserting three repeats of the ATF/CREbinding element into the plasmid pG51uc,replacing the GAL-4 binding *** plasmids pACT/ATF3 andpATF/CRE-luc were transfected into heLa and NIH3T3 cells,respectively,and the results showed that theexpression of luciferase .was increased in a dose-dependent manner on plasmid pACT/*** datasuggested that the plasmid pATF/CRE-luc could be used as a sensitive and convenient reporter system ofATF3 activity.

关键词： ATF/CRE luciferase gene pG51uc reporter system

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Preparation and characterization of solid dispersions of Quercetin with PEG4000

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沈阳药科大学学报 2008年第S1期25卷 129-130页

作者： YANG Xiao-zhan,LI Xiao-hui(Institute of Materia Medica,third military medical university,chongqing 400038,china)

Objective To enhance the solubility,quicken the speed of digesting and absorption,and increase the bioavailability of quercetin(3,3',4',5,7-pentahydroxyflavone).Methods A series of Quercetin-PEG4000 solid dispersions were prepared by fusion *** configuration and property of solid dispersion were characterized by solubility tests,dissolution tests,FTIR spectra,differential scanning calorimetry(DSC)and *** *** to solubility tests the the mass ratio of quercetin to PEG4000 affected strongly on the solubility of solid dispersions,on the whole,the relation of the solubility of solid dispersions to the mass ratio presented linear *** preparation temperature had little effect on the solubility of solid *** surface-active agent,polysorbate80 increased strongly the solubility of solid *** to the dissolution tests,the mass ratio of quercetin to PEG4000 affected strongly on the dissolution of solid dispersions,the preparation temperature had little effect on the dissolution of solid *** surface-active agent,polysorbate80 increased strongly the dissolution of solid dispersions,and after addition polysorbate80,the dissolution of solid dispersions was two times of the dissolution of solid dispersions without *** to the DSC results,except that a little of quercetin molecular existed as crystalline state in the solid dispersion with the mass ratio was qu:PEG=1:2,quercetin existed as amorphous phase in other mass ratio solid *** to the FTIR spectra and microphotograph results,the relation of quercetin and PEG4000 was mainly physical mixing in quercetin-PEG4000 solid *** was just like solute in solution,and PEG4000 was just like solvent in *** force between quercetin and PEG4000 was mainly hydrogen bonding,so the biological activity of quercetin would not be influenced greatly after the formation solid *** Th

关键词： quercetin PEG4000 solid dispersion solubility dissolution

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The ROLE OF PERIAQUEDUCTAL GRAY (PAG) IN The ACUPUNCTURE ANALGESIA AND MODULATION OF PAIN

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针刺研究 1989年第Z1期 87-88页

作者： Zhang Changcheng Li Xicheng Department of Physiology, The third military medical university, chongqing

The role of PAG in the acupuncture analgesia 1. After noxious stimulation of splanchnic nerve or tail, the majority ofunits responded with increased frequency, some of units showed decreased fre-quency and the remainder had no response to both of noxious stimulation. Itseems to imply that PAG is one of the brain structures which receive somati-co-visceral pain impulses.

关键词： PAG IN The ACUPUNCTURE ANALGESIA AND MODULATION OF PAIN The ROLE OF PERIAQUEDUCTAL GRAY

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POTENTIATION EFFECT OF DIAZEPAM ON ACTH ANALGESIA IN The MICE

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针刺研究 1989年第Z1期 117-118页

作者： Li Haiti Zhang Wende Department of Physiology, third military medical university, chongqing

In the present study we injected diazepam, r-aminobutyric acid (GABA)receptor antagonist——picrotoxin and GABA synthesis inhibitor——isoniazid into the intraperitoneal (ip). Mice painful threshold and serotonin (5-HT)content in mice brain were measured by spectrofluorometric assay, the effectof diazepam on ACTH analgesia was obstraeted. The possible mechanisms ofdiazepam effect were studied.

关键词： ACTH POTENTIATION EFFECT OF DIAZEPAM ON ACTH ANALGESIA IN The MICE

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