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Establishment of a Sandwich ELISA Method for Detection of Vascular Endothelial Growth Factor in Serum Samples of Hepatocellular Carcinoma Patients

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Biomedical and Environmental Sciences 2008年第1期21卷 69-74页

作者： BING SHAN CHEN GAO JIAN-MING CHEN XIN-Yu BI BAO-YUN ZHANG YAN GUO CHEN-FANG dong RUN AN QI SHI JING-QUN HU ping ZHAO JUN HAN xiao-ping dong State Key Laboratory for Infectious Disease Prevention and Control National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention Beijing 100052 China Department of Abdominal Surgery Cancer Hospital Chinese Academy of Medical Sciences Beijing 100021 China

Objective To establish a sandwich ELISA method for detecting vascular endothelial growth factor （VEGF） in sera of population and the patients with hepatocellular carcinoma （HCC）. Methods Full length and two truncated human VEGF cDNA sequences were amplified from a commercial plasmid pBLAST49-hVEGF by PCR and inserted into the prokaryotic-expression plasmid pET-32a or pGEX-2T. Various VEGF proteins were expressed and purified from E. coli in His-Trx or GST fusion forms. The specific VEGF antibodies were elicited in experimental rabbits and mice by immunization of the full length VEGF fusion protein His-Trx-VEGF1-165. After purification of antibodies with chromatograph of Protein G, a sandwich ELISA technique was established. Serum VEGF levels were evaluated in 229 adults and 291 HCC patients. Results SDS-PAGE displayed that the molecular weights of the expressed full length （His-Trx-VEGF1-165）, N-terminal （His-Trx-VEGF1-100） and C-terminal （GST-VEGF100-165） human VEGF fusion proteins were about 38KD, 31KD, and 33KD, respectively. Western blots confirmed that the prepared antisera were able to recognize both prokaryoticly and eukaryoticly expressed recombinant VEGF proteins. Assays of serially diluted His-Trx-VEGF1-100 by the established sandwich ELISA method showed that the linear range of the standard curve was 0.625-320 ng/mL, with the squared correlation coefficient R^2=0.991. Screening of a serum panel containing 291 serum samples of HCC patients and 229 health adults revealed that the average VEGF level in HCC patients was higher than that in healthy controls, with a statically significant difference. Conclusion The established sandwich ELISA reflects the level of serum VEGF and provide scientific basis for screening metastasis and recurrence of HCC using serum VEGF as an index.

关键词： Hepatocellular carcinoma Vascular endothelial growth factor Enzyme-linked immunosorbent assay

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Effect of Micro-Area pH Value on Formation of Different Rust Phases of Weathering Steel in Initial Corrosion Period

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Journal of Iron and Steel Research International 2010年第9期17卷 69-72,78页

作者： CHEN xiao-ping WANG Xiang-dong LIU Qing-you MI Feng-yi CHEN Ying LI Yu-su Central Iron and Steel Research Institute Beijing 100081 China

The formation of protective rust layer of atmospheric corrosion resistance was discussed for weathering steel. The rust layers of experimental steels were made by using an accelerating industrial atmospheric corrosion test of dry-wet cyclic immersion. Furthermore XRD were used to measure and analyze the rust layers. The initial corrosion processes below thin water film were observed and the changes of micro-area pH value were measured. The results are as follows： The corrosion processes of P-RE weathering steel are obviously different from those of carbon steel. The surface of P-RE weathering steel rapidly forms a-FeOOH and the micro-area pH value is above 5.4 during the initial corrosion period. While the component of the rust layer for the carbon steel is mainly Fe3O4 and the microarea pH value is below 3.8.

关键词： weathering steel pH value micro area rust phase

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Expressions of Nucleoside Diphosphate Kinase (nm23) in Tumor Tissues are Related with Metastasis and Length of Survival of Patients with Hepatocellular Carcinoma

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Biomedical and Environmental Sciences 2010年第4期23卷 267-272页

作者： RUN AN JIE MENG QI SHI xiao-XIA DAI JING-HONG CHEN YAN-JUN LEI BING SHAN CHEN GAO YONG-LIE CHU xiao-ping dong School of Medicine Xi'an Jiao-Tong University Xi'an 710061 Shanxi China State Key Laboratory for Infectious Disease Prevention and Control National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention Ying-Xin Rd 100 Beijing 100052 China

Objective To evaluate the relationship of expressions of nucleoside diphosphate kinase （nm23） and proliferating cell nuclear antigen （PCNA）, as well as apoptosis, with the prognosis of HCC patients by analyzing their pathological and clinical data. Methods The expressions of nm23 and PCNA were analyzed by immunohistochemistry and the apoptotic phenomena were detected by TUNEL technique in the liver samples from 43 HCC tissues, 39 para-neoplastic tissues, and 10 normal tissues. The mean apoptosis index （AI） and proliferative index （PI） in individual sample were calculated. Results As shown by the detection, 32.6% of carcinomas had negative nm23 signal in tumor tissues, whereas all para-neoplastic and normal tissues had positive nm23. The AI in nm23 positive HCC was significantly higher than that in nm23 negative one, with statistical difference （P0.05）. Furthermore, the expressions of nm23, and the values of AI and PI were contrastively analyzed with some main pathological and clinical data of HCC. It revealed that HCC with extrahepatic metastasis showed remarkable correlation with the negative nm23 （P=0.013） and higher PI values of HCC （P=0.015）. The disease-free survival in HCC patients with negative nm23 expression was significantly poorer than that in patients with positive nm23 expression. Conclusion These data suggest that expressions of nm23 protein in tumor tissues are correlated with occurrences of metastasis and length of survival of the HCC patients, which may be an indicator for their prognosis.

关键词： Hepatocellular carcinoma nm23 PCNA Apoptosis Prognosis

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T188K-Familial Creutzfeldt–Jacob Disease, Predominant Among Chinese, has a Reactive Pattern in CSF RT-QuIC Different from D178N-Fatal Familial Insomnia and E200K-Familial CJD

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Neuroscience Bulletin 2019年第3期35卷 519-521页

作者： Kang xiao Qi Shi Wei Zhou Bao-Yun Zhang Yuan Wang Cao Chen Yue Ma Chen Gao xiao-ping dong State Key Laboratory for Infectious Disease Prevention and Control Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases (Zhejiang University)National Institute for Viral Disease Control and PreventionChinese Center for Disease Control and PreventionBeijing 102206China Center for Global Public Health Chinese Center for Disease Control and PreventionBeijing 102206China

Dear Editor,Human prion diseases consist of sporadic, genetic/familial, and acquired forms. The familial form accounts for 5%-15% of all human prion diseases, including familial Creutzfeldt- Jacob disease (fCJD), Gerstmann-Straussler-Scheinker syndrome, and fatal familial insomnia (FFI)[1-3]. All genetic prion diseases are directly associated with mutations (point-mutation or insertion) in the PRNP gene located on human chromosome 20 and encodes prion protein (PrP). So far,>55 mutations in the PRNP gene have been described [4]. Some PRNP mutations and their related genetic prion diseases have been reported worldwide, while others show clear region- or ethnicity-associated features.

关键词： Human prion diseases sporadic ethnicity-associated features

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Establishment of Cell Free Conversion System With Biotin-labelled Recombinant PrP^(sen) Expressed in ***

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Biomedical and Environmental Sciences 2006年第3期19卷 214-218页

作者： JIN ZHANG JIAN-MEI GAO FENG LI JUN HAN LAN CHEN BAO-YUN ZHANG xiao-FAN WAMG WEI ZHOU YONG LIU xiao-ping dong School of Medicine Xi＇an Jiaotong University Zhuque Street 205 Xi ＇an 710061 China State Key Laboratory for Infectious Disease Prevention and Control National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention Yingxin Rd 100 Beijing 100052 China National Laboratory of Medical Molecular Biology Institute of Basic Medical Science Chinese Academy of Medical Sciences and Peking Union Medical College Dongdan Santiao 5 Beijing 100005 China

Objective To report a protocol using biotin-labelled PrP protein in cell free conversion assay instead of isotope. Methods A hamster PrP protein （HaPrP） was expressed in E. coli and purified with HIS-tag affinity chromatograph. After being labelled with biotin, HaPrP was mixed with PrP^sen preparation from scrapie strain 263K. Results Protease-resistant bands were detected after four-day incubation. Conclusion The new conversion model provides a reliable, easily handling, and environment-friendly method for studies of prion and transmissible spongiform encephalopathies.

关键词： Transmissible spongiform eneephalopathies Prion Biotin Cell free conversion E. coli

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Preparation of Monoclonal Antibodies Against Prion Proteins With Full-length Hamster PrP

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Biomedical and Environmental Sciences 2005年第4期18卷 273-280页

作者： XIN-LI xiao HUI-YING JIANG JIN ZHANG JUN HAN KAI NIE xiao-BO ZHOU YIN-XIA HUANG LAN CHEN WEI ZHOU BAO-YUN ZHANG YONG LIU xiao-ping dong School of Medicine Xi＇an Jiao-Tong University Zhu-Que Street 205 Xi＇an 710061 Shaanxi China National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention Hngxin Rd. 100 Beijing 100052 China National Laboratory of Medical Molecular Biology Institute of Basic Medical Science Chinese Academy of Medical Sciences and Peking Union Medical College Dong Dan San Tiao 5 Beijing 100005 China

To prepare the PrP specific monoclonal antibodies （mAbs） that can be used for the detection of mammalian prions and study of pathogenesis of prion diseases. Methods Several BALB/c mice were immunized with recombinant hamster prion protein （HaPrP）. Three hybridoma cell lines designated as B7, B9, and B10, secreting monoclonal antibodies against HaPrP, were established by hybridoma technique. The mAbs reactivities were evaluated with ELISA, Western blot, and immunohistochemistry. Results The mAbs produced by these cell lines reacted well with different recombinant hamster PrP proteins. Western blot analyses showed that mAbs B7 and B9 reacted with PrP^Sc from the scrapie-infected animals after proteinase K digestion with three glycosylated forms. The mAbs exhibited cross-reactivity with various PrPc from several other mammalian species, including humans and cattles, lmmunohistochemistry assays confirmed that mAbs B7 and B9 could recognize not only extracellular but also intracellular PrPso. Conclusion The mAbs of prion protein are successfully generated by hybridoma technique and can be applied for the diagnosis of prion associated diseases.

关键词： Prions Hamster prion protein Monoclonal antibodies

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Biotransformation of aesculin by human gut bacteria and identification of its metabolites in rat urine

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World Journal of Gastroenterology 2009年第12期15卷 1518-1523页

作者： Wei-Jun Ding Yun Deng Hao Feng Wei-Wei Liu Rong Hu Xiang Li Zhe-Ming Gu xiao-ping dong Chengdu University of Traditional Chinese Medicine Chengdu 610075 Sichuan Province China XenoBiotic Laboratories Inc. 107 Morgan Lane. Plainsboro NJ 08536 United States

AIM： To observe the biotransformation process of a Chinese compound, aesculin, by human gut bacteria, and to identify its metabolites in rat urine.METHODS： Representative human gut bacteria were collected from 20 healthy volunteers, and then utilized in vitro to biotransform aesculin under anaerobic conditions. At 0, 2, 4, 8, 12, 16, 24, 48 and 72 h postincubation, 10 mL of culture medium was collected. Metabolites of aesculin were extracted 3 × from rat urine with methanol and analyzed by HPLC. For in vivo metabolite analysis, aesculetin （100 mg/kg） was administered to rats via stomach gavage, rat urine was collected from 6 to 48 h post-administration, and metabolite analysis was performed by LC/ESI-MS and MS/MS in the positive and negative modes.RESULTS： Human gut bacteria could completely convert aesculin into aesculetin in vitro. The biotransformation process occurred from 8 to 24 h post-incubation, with its highest activity was seen from 8 to 12 h. The in vitro process was much slower than the in vivo process. In contrast to the in vitro model, six aesculetin metabolites were identified in rat urine, including 6-hydroxy-7-glucocoumarin（M1）, 6-hydroxy-7-sulf-coumarin （M2）, 6, 7-digluco-coumarin （M3）, 6-glc-7-gluco-coumarin （M4）, 6-O-methyl-7-gluco-coumarin （MS） and 6-O-methyl-7- sulf-coumarin （M6）. Of which, M2 and M6 were novel metabolites.CONCLUSION： Aesculin can be transferred into aesculetin by human gut bacteria and is further modified by the host in vivo. The diverse metabolites of aesculin may explain its pleiotropic pharmaceutical effects.

关键词： Aesculin Biotransformation Human gutbacteria Rat urine Sulfated derivatives LC/ESI-MS Aseculetin

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The First Chinese Case of Creutzfeldt-Jakob Disease with Mutation of E200K in PRNP

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Biomedical and Environmental Sciences 2010年第2期23卷 158-160页

作者： CHEN GAO QI SHI WEI ZHOU CHAN TIAN HHUI-YING JIANG BAO-YUN ZHANG JUN HAN xiao-ping dong State Key Laboratory for Infectious Disease Prevention and Control National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention Beijing 102206 China

Objective To investigate epidemiological, clinical and genetic features of the first Chinese case of Creutzfeldt-Jakob disease （CJD） with mutation of E2OOK in PRNP. Methods The general epidemiological and clinical data were collected; CSF 14-3-3 protein was analyzed by Western blot; The PRNP was amplified by PCR and analyzed. Results A missense mutation in codon 200 （E200K） of the PRNP was identified in this patient; CSF 14-3-3 protein was positive; sleep disturbance was the initial sign and the other symptoms gradually appeared, including memory loss, dizziness and ataxia. Conclusion The CJD patient who was first reported in China has a missense mutation in codon 200 （E200K） of the PRNP, and the codon 129 is a methionine homozygous genotype.

关键词： CJD PRNP Sleep disturbance 14-3-3

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Treatment of Scrapie Pathogen 263K With Tetracycline Partially Abolishes Protease-resistant Activity in vitro and Reduces Infectivity in vivo

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Biomedical and Environmental Sciences 2007年第3期20卷 198-202页

作者： YAN-JUN GUO JUN HAN HAI-LAN YAO BAO-YUN ZHANG JIAN-MEI GAO JIN ZHANG XIN-LI xiao xiao-FAN WANG WEI-QIN ZHAO DE-XIN WANG xiao-ping dong State Key Laboratory for Infectious Disease Prevention and Control National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention Beijing 100052 China Department of Neurology Beijing Friendship HospitalCapital Medical University Beijing 100050 China

Objective To study the possible effect of tetracycline on protease-resistant activity in vitro and infectivity in vivo of a scrapie strain 263K. Methods Scrapie pathogens were incubated with tetracycline at different concentrations for various periods of time and protease-resistant PrP signals were evaluated with proteinase K-treatment and Western blots. The preparations treated with tetracycline were intracerebrally inoculated into golden hamsters and typical TSE manifestations were noted. PrP^5c in brain tissues of the infected animals was detected by PrP specific Western blot assays. Results Protease-resistant PrP was significantly reduced in or removed from the preparations treated with tetracycline in a dose-dependant manner. Compared with the control group after incubated for 53.75±0.50 days, the preparations treated with 5 mmol/L and 20 mmol/L tetracycline prolonged the incubation time of 61.5±1.73 and 59.5±0.58 days （P〈0.05）. Conclusion Treatment of scrapie pathogen 263K with tetracycline reduces or removes its protease-resistant activity in vitro.

关键词： Scrapie Prion Tetracycline Protease-resistant activity Animal experiment

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Establishment of a Stable PrP^(Sc) Panel from Brain Tissues of Experimental Hamsters with Scrapie Strain 263K

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Biomedical and Environmental Sciences 2009年第2期22卷 151-156页

作者： BAO-YUN ZHANG CHAN TIAN JUN HAN CHEN GAO QI SHI JIAN-MING CHEN HUI-YING JIANG WEI ZHOU AND xiao-ping dong State Key Laboratory for Infectious Disease Prevention and Control National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention Beijing 100052 China

Objective To establish a stable PrP^Sc panel from brain tissues of experimental hamsters infected with scrapie agent 263K for evaluating diagnostic techniques of human and animals＇ prion diseases. Methods Thirty brain tissue samples from hamsters intracerebrally infected with scrapie strain 263K and another 30 samples from normal hamsters were selected to prepare 10%, 1%, and 0.5% brain homogenates, which were aliquoted into stocks. PrP^Sc in each brain homogenate was determined by proteinase K digestions followed by Western blot assay and partially by immunohistochemistry. Stability and glycoforms of PrP^Sc were repeatedly detected by prp^SC-specific Western blots in half a year and 3 years later. Results PrPsc signals were observed in all 10% brain homogenates of infected hamsters. Twenty out of 30 stocks and 19 out of 30 stocks were PrPsc positive in 1% and 0.5% brain homogenatesof infected hamsters, respectively. TWenty-seven out of 30 stocks presented three positive bands in 10% brain homogenates, whereas none of 1% and 0.5% homogenates contained 3 bands. The detection of prpSc-specific signals stored in half a year and 3 years later demonstrated that the ratio of PrPsc positive samples and glycoforms was almost unchanged. All normal hamsters＇ brain homogenates were PrP^Sc negative. Conclusion A PrP^Sc panel of prion disease can be established, which displays reliably stable prp^Sc-specific signals and glycoforms.

关键词： TSE prp^Sc Panel Glycoforms Stability

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