Objective; To express and purify HCA518 protein and prepare its monoclonal antibody ( McAb). Methods: The HCA518 protein was expressed with gene recombinant technique in prokaryotic system and purified with nickel chelate nitrilotriacetic acid(Ni-NTA) affinity chromatography column. Hybridoma cell lines that secreted anti-HCA518 McAb were established by cells fusion and screened by enzyme linked immunosorbent assay( ELISA). The specificity of anti-HCA518 McAb wa3 identified by Western blot assay. The HCA518 protein in tumor cells was stained by immunoflourescence assay. Results: Rcombinant HCA518 protein was expressed with a purity of 98%. Two hybridoma cell lines was selected and anti-HCA518 McAb was purified from mice ascites. The titers of anti HCA518 McAb in ascites were 1?10-4 and 5?10-4 respectively. The antibody belonged to IgG2b subtype and IgM. Anti-HCA518 McAb specifically reacted with recombinant HCA518 protein and tumor cells'nuclear protein (P100). The HCA518 protein was mainly located in cell nucleus. Conclusion: Stable hybridoma cell lines that secreted anti-HCA518 McAb have been established and anti HCA518 McAb was prepared with high specificity. It has important significance for detecting HCA518 protein in tumor tissues and determining malignant proliferation status of tumor cells and predicting its prognosis.

On the background of internationalization for higher education,we have reformed the educational model for bachelor students majoring in public health laboratory science and quarantine.The exploration and pilot study are focused on educational principles,curriculum planning,teaching patterns,experiments and practice,and teachem training.The four teaching strategies have been firstly recommended as follows:laying the strongest foundation of chemistry,expanding quarantine-related curricula for increasing quarantine capability,making full use of strength of clinical laboratory medicine,emphasizing English application.The curricula structure is refined and more social supporting resources have been got to make globalization to cultivate more health inspection and quarantine talents with multidisciplinary knowledge,abilities and strong adaptability.

Objective To express protein transduction domain (PTD)-deletion proline domain (ΔPRD) Foxp3 fusion protein, and to analyze its influence on mixed lymphocyte reaction in mice.Methods We cloned mouse ΔPRD of Foxp3 gene by PCR, and inserted it into pET28a-PTD, pET28a-PTD-eGFP vector, then expressed fusion proteins in E.coli Rosetta (DE3). The fusion proteins were purified and refolded by Profinity IMAC Ni~(2+)-Charged Resin. The expression of fusion proteins was identified by Western blot. Flow cytometry assay was used to detect the effect of PTD-ΔPRD fusion protein to transduce into mouse EL-4 cells. The ability of fusion protein to inhibit the proliferation of EL-4 cells was analyzed by two-way mixed lymphocyte reaction.Results The PTD-ΔPRD fusion proteins were expressed and purified efficiently. Western blot and flow cytometry indicated that PTD-ΔPRD fusion protein was transduced into EL-4 efficiently. Mixed lympocyte reaction assay showed that PTD-ΔPRD fusion protein had the bioactivity to inhibit the proliferation of EL-4 cells.Conclusion The PTD-ΔPRD fusion protein was expressed in E.coli system and could be transduced into cells effectively, suggesting that PTD-ΔPRD fusion protein may be an inhibitor in lymphocytes from mouse spleen.

Objective To investigate the distribution of TH17 cells in peripheral blood of patients with rheumatoid arthritis(RA). Methods Intracelluar flow cytometirc detection of TH17 cells in peripheral blood was establised using PHA or PMA + Ion as stimulators in vitro. Thirty-five active and 30 stable RA pa-tients and sex and age paired healthy controls were enrolled in this study. Results The stimulating effect of PMA + Ion was better than that of PHA alone. Under PMA + Ion stimulation, the percentage of TH17 cells in peripheral blood from RA patients and healthy controls was increased significantly(P<0.05). With or with-out PMA + Ion stimulation, such percentage in active group was higher than that of stable group and both of them were higher than that of heathy controls(P<0.05). The distribution profile of TH1 cells was similar to that of TH17 cells. Conclusion There is a distribution abnormality of TH17 cells in peripheral blood of RA patients, which could be used as a new marker for the assessment of immunological condition in RA.

Objective To investigate the antimicrobial susceptibility of Pseudomonas aeruginosa (P. aeruginosa) isolated from Zhenjiang area to 13 routinely used antibiotics and identify the structure and dissemination of class Ⅰ integron. Methods K-B test was used to determine the resistant rate of 71 strains of P. aeruginosa. DNA template was extracted by boiling method, PCR method was utilized to detect class Ⅰintegron, and subsequently gene cassettes were analyzed by sequencing. Results The resistant rates to 13 routinely used antibiotics were quite different from 18. 3 to 77.5% among 71 strains of P. aeruginosa. The prevalence of class Ⅰ integron was 38%. These integrons include 5 gene cassettes ( aadB, aac (6) - Ⅱ , PSE-Ⅰ , dfrA17 and aadAS), in which dfrA17 and aadA5 gene cassette were frequently found. Comparing with the negative strains of integron, the positive strains of integron has obviously higher resistance to ten the antibiotics including piporacillin, piperacillin-tazobactam, ceftriaxone, cefepime, ceftazidime, gentamicin,amikacin, tobmmycin, levofloxacin, and ciprofloxacin. Conclusions The resistant rates of P. aeruginosa to 13 drugs were different, and the resistant rates of integron positive strains were obviously higher than integron negative strains, which indicates that integron may play an important role in multidrug reisistance of P. aeruginoosa.

Objective To detect the expression levels of high mobility group box chromosomal protein 1 (HMGB1) and Th17 cells transcription factors, related cytokines in peripheral blood of rheumatoid arthritis (RA) patients and analyze the relations between HMGB1 and CRP, ESR, RF in RA patients. The other aim of this study is to identify the expression level of HMGBI and the relationship between HMGB1 and Th17 in RA patients. Methods The mRNA levels of HMGB1, RORyt, interleukin (IL)-17 in the peripheral blood mononuclear cells (PBMC) were determined by quantitative real-time PCR (QRT-PCR) from 80 patients with rheumatoid arthritis,including 32 RA patients in stable phase and 48 patients in active phase, and 50 healthy volunteers. The concentration of HMGB1, IL-23, IL-17 in plasma were detected by enzyme linked immunosorbent assay (ELISA), one-way ANOVA and Spearman's correleation were adopted for statistical analysis.Results The mRNAs of HMGBI, RORyt and IL-17 in RA patients were higher than that in healthy control group (P<0.05), especially in active RA patients [ HMGB 1 (0.424±0.262) pg/ml, RORγt (0.34±0.25) pg/ml,IL-17 (1.42±0.38) pg/ml,P<0.01 ] when compared with patients with stable disease. The concentration of HMGB1, IL-23 and IL-17 in the plasma of RA patients was higher than that of the healthy control group (P< 0.05), and was positively correlated with the expression levels of HMGB1, Th 17-associated factors and the level of CRP, ESR, RF in RA patients' plasma(P<0.05). Conclusion The HMGB1 and Thl7 cells levels are higher in active RA patients than those in patients with stable disease, arid there is significant positive correlation between them. Detection of peripheral HMGB1 and Thl7 cell-specific transcription factors or related cytokines can help to understand the development and progress of rheumatoid arthritis and provide clues for new treatment targets for RA.

Objective:To investigate the effect of butyrate produced by bacterial metabolism on immune features of murine bone marrow-derived dendritic cells(BMDCs) and its potential mechanisms.Methods: BMDCs were prepared from bone marrow cells of C57BL/6 mice by being cultured with GM-CSF and IL-4.The expression of CD80,CD86,B7-DC and MHCⅡ on BMDCs and its pri-ming ability on the proliferation of OVA257-264 antigen specific CD8+T cell were analyzed by using Flow cytometry.The mRNA levels of IL-6 and IL-12 in BMDCs were detected by real-time fluorescence quantitative PCR(q-PCR).Simultaneously,Griess reaction and Western blot was used for analyzing the levels of NO2-in BMDCs culture supernatant and the ERK phosphortylation in BMDCs respectivly.Results:Butyrate could decrease the levels of CD80,CD86,MHCⅡand B7-DC,and downregulate the capability of BMDCs in priming the proliferation of CD8+T cells.Furthermore,the secretions of IL-6,IL-12,NO2-and the phosphorylation of ERK were sup-pressed.Conclusion:Butyrate down-regulats the immune functions of BMDCs via inhibition of ERK phosphorylation in TLR 4 signaling pathway.

In order to investigate the effect of T-bet on malignant cells, we selected SGC-7901, a kind of human gastric carcinoma cell line, and used gene clone technique and adeno-associated virus (AAV) packing technology, thus obtaining a recombinant rAAV-eGFP-T-bet and T-bet gene-transfected SGC-7901 cells. Then the function of T-bet gene-infected SGC-7901 cells was researched by detecting the levels of IFN-gamma and T-bet production. The results showed: (1) It was verified that rAAV-T-bet's packing was completed; (2) After SGC-7901 cells was transfected by rAAV-eGFP-T-bet, a green fluorescence was found in about 30%-40% SGC-7901s, and the gene of 1670 bp (T-bet) and 388 bp (IFN-gamma) were generated from SGC-7901s cells; (3) The proteins of IFN-gamma and T-bet secreted by SGC-7901 cells were also detected. These reveal that SGC-7901 cell is efficiently infected by rAAV encoding T-bet, which can induce transfected cells to secret IFN-gamma. It may be useful in the researches on cancer immune therapy of transfecting T-bet gene.
Cell Line, Tumor ; Dependovirus ; genetics ; metabolism ; Green Fluorescent Proteins ; biosynthesis ; Humans ; Interferon-gamma ; biosynthesis ; Recombinant Proteins ; biosynthesis ; genetics ; Stomach Neoplasms ; genetics ; metabolism ; T-Box Domain Proteins ; biosynthesis ; genetics ; Transfection

OBJECTIVETo investigate the effect of occupational exposure to toluene diisocyanate (TDI) on the workers' health.

METHODSA total of 76 workers exposed to TDI (exposure group) and 64 management staff members (control group) were selected from a factory as the study subjects. Area sampling was performed for the place with exposure to TDI according to the method in GBZ 159-2004 Specifications of air sampling for hazardous substances monitoring in the workplace, and gas chromatography was applied to measure the concentration of TDI in workplace air. The workers' personal information was collected with questionnaire, pulmonary ventilation function was determined with a portable spirometer, hematological parameters were analyzed by automatic blood analyzer and blood chemistry analyzer, and the indicators of oxidative damage and energy metabolism were measured by the reagent kit provided by Nanjing Jiancheng Bioengineering Institute. SPSS 17 software was applied for statistical analysis.

RESULTSThe exposure group had significantly lower forced vital capacity (FVC), forced expiratory volume in 1 second(FEV1.0), and FEV1.0/FVC ratio than the control group (P <0.05). Compared with the control group, the exposure group had significantly higher red blood cell count, platelet distribution width, mean platelet volume, lymphocyte count, and neutrophil count(P<0.01), and significantly lower activities of lactate dehydrogenase(LDH), superoxide dismutase, and succinodehydrogenase (SDH)(P <0.01). In the exposure group, the length of exposure was negatively correlated with the activities of SDH and LDH in the serum (r=-0.319, P <0.05; r=-0.239, P <0.05), and the length of exposure was not found to be correlated with the activity of SOD and pulmonary function indices.

CONCLUSIONTDI can induce inflammatory response and lung ventilation function impairment in workers exposed to TDI, as well as oxidative stress and imbalance of energy metabolism. Therefore, it can cause damage to workers' health, and protective measures should be enhanced.

Case-Control Studies ; Erythrocyte Count ; Forced Expiratory Volume ; Humans ; Inflammation ; physiopathology ; L-Lactate Dehydrogenase ; blood ; metabolism ; Leukocyte Count ; Lung ; physiopathology ; Occupational Exposure ; adverse effects ; Pulmonary Ventilation ; Succinate Dehydrogenase ; blood ; metabolism ; Superoxide Dismutase ; metabolism ; Toluene 2,4-Diisocyanate ; adverse effects ; Vital Capacity

Objective To Analyze the expression profiles of LncRNAs and mRNAs in ovarian epithelial cancer cell lines by gene mi-croarray, and then provide experimental evidences for investigating the function of LncRNAs associated with ovarian cancer. Methods The differentially expressed LncRNAs and mRNAs in ovarian epithelial cancer cell lines, such as A2780, HO8910 and SKOV3, and ovarian epithelial cell line HOSEpiC were analyzed by gene microarray. The differentially expressed mRNAs were further performed the KEGG pathway enrichment analysis. The expression levels of six candidate LncRNAs, which had significant difference between the o-varian epithelial cancer cell line and the ovarian epithelial cell line, were further verified by qRT-PCR. Results There were 227 up-regulated LncRNAs and 483 down-regulated LncRNAs in A2780, HO8910 and SKOV3 cell lines. The differentially expressed mRNAs in A2780, HO8910 and SKOV3 cell lines were mainly enriched in the tumor-related pathways such as PI3K-AKT, mTOR and TNF-α( P<0.05) . The expression levels of PTPRG-AS1, CCNT2-AS1, XLOC 009869 and LINC01138 in ovarian epithelial cancer A2780, SKOV3 and OVCR3 cell lines were up-regulated (P<0.05), while those of RP11-252P19.2 and RP11-744I24.2 in ovarian epithelial cancer A2780, SKOV3, OVCR3 and 3AO cell lines were down-regulated ( P<0.05) . Conclusion The differentially expressed LncR-NAs and mRNAs in ovarian epithelial cancer cell lines may be obtained by gene microarray, and the differentially expressed mRNAs are associated with the tumor-related pathways such as PI3K-AKT, mTOR and TNF-α, which may provide new targets for the diagnosis and treatment of ovarian cancer.