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Stable Skin-specific Overexpression of Human CTLA4-Ig in Transgenic Mice through Seven Generations

Acta Biochimica et Biophysica Sinica 2006年第3期38卷 171-178页

作者： Yong WANG~1 Yong NI~(1,3) Hong WEI~1* Feng-Chao WANG~2 Liang-Peng GE~1 Xiang GAO~41 Department of Animal Science,college of Basic medicine,third military medical university,chongqing 400038,china 2 institute of combined injury,college of military preventive medicine,third military medical university,chongqing 400038,china 3 School of Life Sciences,Southwest university,chongqing 400715,china 4 Animal Model Research institute,Nanjing university,Nanjing 210093,china Department of Animal Science College of Basic Medicine Third Military Medical UniversityChongqing 400038 China School of Life Sciences Southwest UniversityChongqing 400715 China institute of combined injury College of Military Preventive Medicine Third Military Medical UniversityChongqing 400038 China Animal Model Research institute Nanjing UniversityNanjing 210093 China

Skin graft rejection is a typical cellular immune response,mainly mediated by T *** lymphocyte associated antigen 4-immunoglobin (CTLA4-Ig) extends graft survival by blocking the T cellco-stimulation pathway and inhibiting T cell *** investigate the efficacy of CTLA4-Ig in prolong-ing skin graft survival,human CTLA4-Ig (hCTLA4-Ig) was engineered to overexpress in mouse skin bytransgenesis using the K14 *** transcription-polymerase chain reaction (RT-PCR) and West-ern blot assay indicated that the expression of CTLA4-Ig remained skin-specific and relatively constantcompared to the internal control protein,AKT,through seven *** presence and concentrationof the hCTLA4-Ig protein in transgenic mouse sera was determined by enzyme-linked immunosorbent assay(ELISA),and the results indicated that the serum CTLA4-Ig concentration also remained constant *** of transgenic mouse skins grafted onto rat wounds was remarkably prolonged com-pared to that of wild-type skins from the same mouse strain,and remained comparable among all *** suggested that the bioactive hCTLA4-Ig protein was stably expressed in transgenical micethrough at least seven generations,which was consistent with the stable skin-specific CTLA4-Ig *** results demonstrated that the transgenic expression of hCTLA4-Ig in skin driven by the K14 promoterremained constant through generations,and a transgenic line can be established to provide transgenic skinwith extended survival reproducibly.

关键词： cytotoxic T lymphocyte antigen 4-Ig transgenic mice skin K14 promoter

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Influencing factors of rat small intestinal epithelial cell cultivation and effects of radiation on cell proliferation

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World Journal of Gastroenterology 2001年第1期7卷 140-142页

作者： Xin Ze Ran Yong Ping Su Yong Jiang Wei Guo Ping Ai Tian Min Cheng Yuan Lin institute of combined injury, PLA, School of preventive medicine, third military medical university. chongqing 400038, china institute of combined injury PLA School of Preventive Medicine Third Military Medical University Chongqing 400038 China. rxz@***

INTRODUCTIONCrypt epithelial cells in normal small intestineproliferate at a high speed. But they are verydifficult to culture in vitro and passage stably. A lotof studies have been done[1-16]. Some domestic labsisolated and cultured crypt cells from embryonalintestines and aseptic animal intestine, but *** introduced normal rat epithelial cell line-IEC-6from the USA and its living condition for stablepassage was successfully established after trials. Thecell line was testified to be the small intestinalepithelial cell by electron microscopy,immunihistochemistry and enzymatic histoch-emistry. It has been applied to some relatedresearch work[17-21]. It was found that manyfactors were involved in the culture system. Ourpresent study focuses on the culture method and theinfluencing factors on IEC-6.

关键词： Animals Blood Proteins Cell Count Cell Culture Techniques Cell Division Cell Line Culture Media Dose-Response Relationship, Radiation Fetal Proteins Hydrogen-Ion Concentration Hypoglycemic Agents Insulin Intestinal Mucosa Intestine, Small Radiation, Ionizing Rats Reproducibility of Results Research Support, Non-U.S. Gov't Thymidine Tritium

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Differentially expressed proteins of gamma-ray irradiated mouse intestinal epithelial cells by two-dimensional electrophoresis and MALDI-TOF mass spectrometry

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World Journal of Gastroenterology 2003年第12期9卷 2726-2731页

作者： Bo Zhang Yong-Ping Su Guo-Ping Ai Xiao-Hong Liu Feng-Chao Wang Tian-Min Cheng institute of combined injury of PLA,third military medical university,chongqing 400038,china institute of combined injury of PLA Third Military Medical University Chongqing 400038 China.

AIM: To identify the differentially expressed proteins involved in ionizing radiation in mice and to explore new ways for studying radiation-related ***: Bal B/c mice grouped as sham-irradiation, 3h and 72 h irradiation were exposed to 9.0Gy single dose of γ-irradiation. Intestinal epithelia were isolated from mice,and total proteins were extracted with urea containing solution. A series of methods were used, including twodimensional electrophoresis, PDQuest 2-DE software analysis, peptide mass fingerprinting based on matrixassisted laser desorption/ionization time of flight masss pectrometry (MALDI-TOF-MS) and SWISS-PROT database searching, to separate and identify the differential *** blotting and RT-PCR were used to validate the differentially expressed ***: Mouse intestine was severely damaged by 9.0 Gy γ-irradiation. Image analysis of two-dimensional gels revealed that averages of 638±39, 566±32 and 591±29 protein spots were detected in 3 groups, respectively, and the majority of these protein spots were matched. About 360 protein spots were matched between normal group and 3 h irradiation group, and the correlation coefficient was 0.78 by correlation analysis of gels. Also 312 protein spots matched between normal group and 72 h irradiation group, and 282 protein spots between 3 h and 72 h irradiation groups. Twenty-eight differential protein spots were isolated from gels, digested with trypsin, and measured with MALDI-TOF-MS. A total of 25 spots yielded good spectra, and 19 spots matched known proteins after database searching. These proteins were mainly involved in anti-oxidation, metabolism, signal transduction,and protein post-translational processes. Western-blotting confirmed that enolase was up-regulated by γ-irradiation. Up-regulation of peroxiredoxin I was verified by applying RTPCR technique, but no change occurred in ***: These differentially expressed proteins might play important roles when mouse

关键词： MALDI-TOF-MS 二维电泳上皮细胞 γ射线动物实验放射损伤

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Prenatal exposure to lipopolysaccharide results in increases in blood pressure and body weight in rats

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Acta Pharmacologica Sinica 2007年第5期28卷 651-656页

作者： Yan-ling WEI Xiao-hui LI Jian-zhi ZHOU institute of Materia Medica,School of Basic medicine,third military medical university,chongqing 400038,china institute of Materia Medica School of Basic Medicine Third Military Medical University Chongqing China

Aim:To investigate the effects of prenatal exposure to lipopolysaccharide (LPS)on blood pressure and body weight of offspring in ***:Sixteen healthy,pregnant rats were randomly divided into 2 *** rats in the LPS groupwere injected intraperitoneally with LPS (0.79 mg/kg) on the d 8,d 10,and d 12 *** in the control group were only treated with normal ***,all offspring were weighed and blood pressure was measured by the tail-cuff method once every 2 weeks from the 6th to the 24th *** the 15th week,their food intake was weighed every *** the end of the 24th week,the ratswere killed by *** adipose tissues were weighed,and theserum level of leptin was detected by ***:The offspringwith prenatal LPS exposure showed increased systemic arterial pressure,heavierbody weight,elevated food intake,increased adipose tissue weight,and increasedcirculating leptin compared with the ***:Prenatal exposure toLPS leads to increases in blood pressure and body weight in rats.

关键词： hypertension inflammation obesity prenatal exposure

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Analysis of spontaneous, gamma ray-and ethylnitrosourea-induced hprt mutants in HL-60 cells with multiplex PCR

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World Journal of Gastroenterology 2003年第3期9卷 578-583页

作者： Sheng-Xue Liu Jia Cao Hui An Hua-Min Shun Lu-Jun Yang Yong Liu Department of Health Toxicology,preventive medical college,third military medical university,chongqing 400038,china Department of Healath Toxicology Preventive Medical College Third Military Medical University Chongqing 400038 China.

AIM: To explore the molecular spectra and mechanism of human hypoxanthine guanine phosphoribosyl transferase (hprt) gene mutation induced by ethyluitrosourea (ENU) and 60Co γ-***: Independent human promyelocytic leukemia cells (HL-60) mutants at the hprt locus were isolated from untreated, ethyluitrosourea (ENU) and 60Co γ-ray-exposed cells, respectively, and verified by two-way screening. The genetic changes underlying the mutation were determined by multiplex polymerase chain reaction (PCR) amplification and electrophoresis ***: With dosage increased, survival rate of plated cell reduced (in the group with dosage of ENU with 100-200μg/ml, P＜0.01; in the group with dosage of 60Co γ-ray with 2-4 Gy, P＜0.05) and mutational frequency increased (in the group of ENU 12.5-200.0 μg/ml, P＜0.05; in the group of 60Co γ-ray with 1-4 Gy, P＜0.05) significantly. In the 13spontaneous mutants analyzed, 92.3 % of mutant clones did not show any change in number or size of exon, a single exon was lost in 7.7 %, and no evidence indicated total gene deletion occurred in nine hprt exons. However, deletions were found in 79.7 % of ENU-induced mutations (62.5-89.4 %,P＜0.01) and in 61.7 % of gamma-ray-induced mutations (28.6-76.5 %, P＜0.01). There were deletion mutations in all 9 exons of hprt gene and the most of induced mutations were chain deletion with multiplex exons (97.9 % in gammaray-induced mutants, 88.1% in ENU-induced mutants).CONCLUSION: The spectra of spontaneous mutations differs completely from that induced by EUN or 60Co γ-*** both ENU and γ-ray can cause destruction of genetic structure, mechanism of mutagenesis between them may be different.

关键词：人次黄嘌呤鸟嘌呤转磷酸糖基酶 HL-60细胞 γ射线多重PCR 基因突变分子生物学

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Mechanical properties of hepatocellular carcinoma cells

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World Journal of Gastroenterology 2002年第2期8卷 243-246页

作者： Gang Zhang,Department of Pathophysiology,The third military medical university,chongqing 400038,china Mian Long Zhe-Zhi Wu Wei-Qun Yu,college of Bioengineer,chongqing university,chongqing 400044,china Department of Pathophysiology The Third Military Medical University Chongqing 400038 China. a65412423@***

AIM: To study the viscoelastic properties of human hepatocytes and hepatocellular carcinoma (HCC) cells under cytoskeletal perturbation, and to further to study the viscoelastic properties and the adhesive properties of mouse hepatoma cells (HTC) in different cell cycle. METHODS: Micropipette aspiration technique was adopted to measure viscoelastic coefficients and adhesion force to collagen coated surface of the cells. Three kinds of cytoskeleton perturbing agents, colchicines (Col), cytochalasin D (CD) and vinblastine (VBL), were used to treat HCC cells and hepatocytes and the effects of these treatment on cell viscoelastic coefficients were investigated. The experimental results were analyzed with a three-element standard linear solid. Further, the viscoelastic properties of HTC cells and the adhesion force of different cycle HTC cells were also investigated. The synchronous G(1) and S phase cells were achieved through thymine-2-desoryriboside and colchicines sequential blockage method and thymine-2-desoryriboside blockage method respectively. RESULTS: The elastic coefficients, but not viscous coefficient of HCC cells (K(1)=103.6+/-12.6N.m(-2), K(2)=42.5 +/ 10.4N.m(-2), mu=4.5 +/- 1.9Pa.s), were significantly higher than the corresponding value for hepatocytes (K(1)=87.5 +/- 12.1N.m(-2), K(2)=33.3+/-10.3N.m(-2), mu=5.9+/-3.0Pa.s, P2) to 81.7N.m(-2), 66.3% to 78.9%; K(2): 34.5N.m(-2) to 37.1N.m(-2), 81.2% to 87.3%, P2.6N.m(-2) to 49.8N.m(-2), 48.7% to 56.9%; K(2): 17.2N.m(-2) to 20.4N.m(-2), 51.7% to 61.3%, P2.0 to 3.4Pa.s, 44.4 to 75.6%, P<0.001) than that for hepatocytes (3.0 to 3.9Pa.s, 50.8 to 66.1% P<0.001).

关键词： Animals Antineoplastic Agents, Phytogenic Carcinoma, Hepatocellular Cell Adhesion Cell Cycle Colchicine Cytochalasin D Cytoskeleton Elasticity Hepatocytes Humans Liver Neoplasms Mice Nucleic Acid Synthesis Inhibitors Research Support, Non-U.S. Gov't Tumor Cells, Cultured Vinblastine

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Construction and characterization of an experimental ISCOMS-based hepatitis B polypeptide vaccine

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World Journal of Gastroenterology 2002年第2期8卷 294-297页

作者： Xiao-Ju Guan Shanghai institute of Materia Medica,Chinese Academy of Sciences,Shanghai 200031,china,previously worked as a postdoc in institute of Immunology,third military medical university,chongqing 400038,china Xiao-Jun Guan,Department of Science & Research,Second military medical university,Shanghai 200433,china Yu-Zhang Wu Zheng-Cai Jia Tong-Dong Shi Yan Tan,institute of Immunology,third military medical university,chongqing 400038,china Shanghai institute of Materia Medica Chinese Academy of Sciences Shanghai 200031 China.

AIM: To characterize the biochemical and immunological properties of an experimental ISCOMS vaccine prepared from a novel therapeutic polypeptide based on T cell epitopes of HBsAg, and a heptatis B-ISCOMS was prepared and investigated. METHODS: An immunostimulating complexes(ISCOMS)-based vaccine containing a novel therapeutic hepatitis B polypeptide was prepared by dialysis method, and its formation was visualized by electron microscopy and biochemically verified by SDS-polyacrylamide gel electrophoresis. Amount of the peptide within ISCOMS was determined by Bradford assay, and specific CTL response was detected by ELISPOT assay. RESULTS: Typical cage-like structures of submicroparticle with a diameter of about 40nm were observed by electron microscopy. Results from Bradford assay showed that the level of peptide incorporation was about 0.33g.L(-1). At the paralleled position close to the sixth band of the molecular weight marker(3480kDa) a clear band was shown in SDS-PAGE analysis, indicating successful incorporation of polypeptide into ISCOMS. It is suggested that ISCOMS delivery system could efficiently improve the immunogenicity of polypeptide and elicit specific immune responses in vivo by the results of ELISPOT assay, which showed that IFN-gamma producing cells(specific CTL responses) were increased(spots of ISCOMS-treated group: 47+/-5, n =3; control group: 5+/-2, n =3). CONCLUSION: ISCOMS-based hepatitis B polypeptide vaccine is successfully constructed and it induces a higher CTL response compared with short polypeptides vaccine in vivo.

关键词： Hepatitis B Vaccines ISCOMs Animals Enzyme-Linked Immunosorbent Assay Epitopes Female Hepatitis B Surface Antigens Humans Interferon Type II Mice Mice, Inbred BALB C Peptides Research Support, Non-U.S. Gov't T-Lymphocytes, Cytotoxic

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Cytotoxic T lymphocyte response induced by an improved synthetic lipopeptide vaccine against cervical cancer

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Acta Pharmacologica Sinica 2007年第5期28卷 695-702页

作者： Dao-hua XU~(1,5),Chen-hui ZHOU~2,Yong-peng XIA~3,Zong-ying QIU~3,Yu-zhang WU~4,Zheng-cai JIA~4,Wei ZHOU~4~1 Department of Pharmacology and ~2 School of Nursing,Guangdong medical college,Zhanjiang 524023,china ~3 Department of Pharmacychongqing university of medical Science,chongqing 400016,china,~4 institute of Immunology PLA,third military medical university,chongqing 400038,china Department of Pharmacology Guangdong Medical College Zhanjiang China School of Nursing Guangdong Medical College Zhanjiang China Department of Pharmacy Chongqing University of Medical Science Chongqing China institute of Immunology PLA Third Military Medical University Chongqing China

Aim:To explore cytotoxic T lymphocyte （CTL） response induced by the lipopeptidevaccine against cervical ***:The immunological effect inducingCDS+ T cell-mediated cytotoxicity was investigated in human leukocyte antigen（HLA）-A2 transgenic mice and peripheral blood mononuclear cells （PBMC） ofhealthy HLA-A2.1+blood *** activity of specific CTL was measured byusing a standard 4 h51Cr release *** content of major histocompatibilitycomplex （MHC） I on T2 cells and the expression of immune molecules on dendriticcells （DC） were detected by flow cytometry,and the concentrations of interleukin（IL）-12 and interferon-γ were determined by ***:The lipopeptideinduced a strong epitope-specific CTL response both in vivo （transgenic mice）and in vitro （human PBMC）.This CTL induction was critically dependent on thepresence of the helper T lymphocyte epitope in transgenic mice,and the presenceof a lipid tail bypassed the need for an *** stability and persistence ofthe antigenic complex formed with the lipopeptide increased in comparison withthe CTL parental *** lipopeptide could induce the production of IL-12 inDC,but not the maturation of DC ***:The combination of CTLand the T helper epitope and lipid molecule can remarkably improve the immuno-genicity of the CTL peptide,the mechanism of which is associated with an in-crease in the stability and persistence of the antigenic complex formed with thelipopeptide and in the production of IL-12 in DC induced by the *** can be considered a more effective vaccine type for human being.

关键词： epitope cervical cancer lipopeptide vaccine

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Anti-inflammatory effects of a novel peptide designed to bind with NF-κB p50 subunit

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Acta Pharmacologica Sinica 2006年第11期27卷 1474-1478页

作者： Zhen-ping WANG~2 Shao-xi CAI~(2,4)Dong-bo LIU~3 Xiang XU~3 Hua-ping LIANG~3 2 Bioengineering college,chongqing university,chongqing 400044,china 3 State Key Laboratory of Trauma,Burns and combined injury,Department 1,Research institute of Surgery,Daping Hospital,third military medical university,chongqing 400042,china Bioengineering college Chongqing University Chongqing China Department 1 State Key Laboratory of Trauma Burns and Combined Injury Research Institute of Surgery Daping Hospital Third Military Medical University Chongqing China

Aim:To explore the anti-inflammatory effects of a novel peptide designed to bindwith the NF-κB p50 ***:The affinity of the peptide binding withp50 was measured with a *** of tumor necrosis factor-α(TNF-α)and interleukin 6(IL-6)from a human acute monocytic leukemia cell line(THP-1)treated with lipopolysaccharide(LPS)were measured using the ELISA method,Invivo anti-inflammatory effects of the peptide were tested with phorbol myristateacetate(PMA)-induced ear edema and zymosan A-induced peritonitis in ***:The peptide has the ability to interact with the NF-κB p50 subunit andcan effectively inhibit TNF-α and IL-6 production in the THP-1 cell line,PMA-induced ear edema and zymosan A-induced peritonitis in ***:Thepeptide may have therapeutic potential for the treatment of local acute inflammation.

关键词： NF-kB anti-inflammatory agents cytokines

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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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