Objective To investigate the effects of osteopontin (OPN) antisense oligodeoxynucleotide (AS-ODN)on OPN mRNA expressions and adherence to collagen gel of rat renal mesangial cell line (1097). Methods One AS-ODN complementary chain to rat OPN cDNA sequences was designed and synthesized. All nucleotides of different kinds of oligodeoxynucleotide (including antisense, sense and mismatch sense) were modified with phosphrothioate. All the ODNs were mixed with cationic liposome(DOTAP) respectively. The cultured 1097 cells were incubated with different concentration of ODN at 37t for 48 hours, then total RNA was extracted from cultured cell line with Trizol reagent. OPN mRNA expression was detected with RNA dot blot and RT-PCR. The cell adhesion ability was measured with collagen gel attachment lest. Results OPN mRNA expression of mesangial cells was higherly upregulated in the medium with calf serum compare to that in the medium without serum. AS-ODN could suppress the expression of OPN mRNA in mesangial cells with dose-dependent and its lowest inhibitory concentration was 2. 5 ?mol/L, but mismatch ODN or sense ODN have no inhibitory effects on the OPN mRNA expression of mesangial cells even if at a high ODN concentration of 30 ?mmol/L. AS-ODN-treated cells weakly adhered to the collagen gel and could be easily detached off. The mesangial cells treated with mismatch ODN or sense ODN still had a good adherent ability to collagen gel. Conclusions Calf serum can induce rat renal mesangial cells higherly expressing OPN mRNA. Osteopontin antisense oligodeoxynucleotide can specifically suppress mesangial cells OPN mRNA expression and adhension to collagen gel.

Objective To clone and express Mycobacterium tuberculosis fusion antigen ESAT-6 and CFP-10 ( EC) and to evaluate the biological activity of the fusion antigen EC in inducing specific cytokines secretion from THP -1 cells. Methods The fusion antigen EC gene was cloned into pET-30 a prokaryotic expression vector and expressed highly in E.coli BL21.Then, the THP-1 cells were stimulated with purified fusion antigen EC of different concentrations (10 and 20 μg/ml).Culture supernatants were collected after 12 h and 24 h, respectively.The secretion levels of IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, TNF-αand IFN-γin THP-1 cell culture supernatants were detected using Bio-PlexProTM Assays kit.Results The M.tuberculosis fusion antigen EC was cloned and expressed successfully .The secretion levels of IL-6, IL-8 and TNF-αin EC infected THP-1 cells were significantly higher than those in THP-1 cells (P<0.05).The secretion levels of other cytokines did not change significantly .Conclusion The obtained M.tuberculosis fusion antigen EC has biological activity in inducing the THP-1 cells to secrete a higher level of IL-6,IL-8 and TNF-α.

Objective:To research the solubilization of sulfobutyl ether-β-cyclodextrin ( SEB-β-CD) on voriconazole and the sta-bility of voriconazole with different pH values. Methods:Phase-solubility diagram was applied to study the solubilization of SEB-β-CD on voriconazole. Meanwhile, the impurity of voriconazole was determined to study the stability. Results:SBE-β-CD could increase the solubility of voriconazole significantly and the apparent stability constant was similar within the pH range of 5. 0-8. 0. The stability of voriconazole was decreased with the increase of pH. Conclusion:SEB-β-CD is an ideal solubilizer for voriconazole, while the stability is influenced by pH.

Objective To develop an antigen retrieval method for detection of human mammaglobin ( hMAM) immuno-histochemcal staining in old paraffin-embedded specimens .Methods The tissue sections in test group were put into dis-tilled water after deparaffinization and then moved into citric acid buffer ( pH 3.5) for 10-15 min.The other two meth-ods,microwave method and high pressure cooker method ,were compared as control groups at the same time .Finally, immu-nohistochemistry SP method was used to check the antibody in the sections .Results The color appearance in the test group (pH 3.5 citric solution) was better than that of microwave oven and high pressure cooker groups .In the test group, tissue sections were not easily cast off from the slices .Conclusion In this study,we have established a new and simple antigen retrieval method which will contribute to immunohistochemistry technology .

Objective To provide the candidate antigens for immunological diagnosis by analyzing the expression of nu -cleoprotein ( NP) of Ebola virus. Methods BioSun software was used to predict the NP epitopes. The bridging-PCR was used to synthesize the NP gene. The pBVIL1 vector was used to clone and express the NP gene. Results The 360-739 aa of NP was confirmed to be the dominant antigen by BioSun software. The recombinant NP dominant antigen was expressed in E.coli with molecular weight of 58 ×103.The specificity of ELISA based on recombinant NP was 99.24% (130/131) in negative samples. Conclusions The dominant NP antigen can be potentially used for developing Ebola virus diagnostic reagent.

Objective To research CpG and Al(OH)3 adjuvants enhancing immunogenicity of hepatitis C virus(HCV) recombinant ptotein combined vaccine(TIE).Methods BALB/c mice were immunized with candidate vaccine TFE using CpG,Al(OH)3,Al(OH) 3 + CpG,or freund's adjuvant(FA) as the adjuvant.Five mice were sacrificed after 10 d of the last immunization.Specific antibodies in sera were tested by enzyme-linked immunosorbent assay(ELISA).Splenic cells were isolated and levels of IFN-γ,IL-4 and cytotoxic T lymphocyte(CTL) cytotoxicity assay were messuredin vitro.The remaining mice were subcutaneouly injected with 1 × 106 SP2/0-NS3 cells on the back to investigate the protective effects.The differences of means between groups were compared by LSD-t test.Results The specific CTL activity of TFE + A1(OH) 3 + CpG group was higher than TFE + FA group and TFE + CpG group(P ＜ 0.05).The level of IFN-γsecreting cells in TFE + Al(OH)3 + CpG group was higher than that in TFE + M(OH)3 group or TFE + CpG group(P ＜ 0.05).Conclusion Combining Al(OH) 3 and CpG could enhance specific cellular immunogenicity of candidate HCV vaccine TFE.TFE + M(OH) 3 + CpG could effetively prevent the attack of tumor cell SP2/0-NS3 expressing nonstructural protein NS3 of HCV.

Objective To explore the relationship between HBV-DNA load and the offspring vertical transmission of HBV.Methods 138 families who had taken the examination between August 2009 and November 2011 but the HBsAg of the housewife was negative,were chosen as research objects.Blood from the couples and sperms from the husbands during pregnancy were followed and collected for detection on related indicators.Cord blood was sampled after delivery for HBVM and HBV-DNA quantification.Those with HBV-DNA load ≥5 × 102 copies/ml were chosen as cases while those ＜5 × 102 copies/ml were formed as controls,respectively.Results 1) The positive rates of HBV-DNA was 34.8％ (48/138) in the neonatal cord blood while the positive rates of cord blood HBsAg and HBeAg were 28.3％ (39/138) and 15.2％ (21/138) respectively.2) The positive rate of semen HBV-DNA was 21.0％ (29/138) while the positive rates of paternal serum HBV-DNA and HBeAg were 76.8％ (106/138)and 42.8％ (59/138).3)Among the positive ones on paternal serum HBV-DNA,paternal serum HBeAg,semen HBV-DNA,items as measures taken for HBV vertical transmission and prevention on the fathers and the first class family histories on HBV appeared to be the risk factors for HBV paternal transmission (P＜0.05).4)Data from Multivariate analysis showed that positivities on patemal serum HBV-DNA,paternal serum HBeAg and semen HBV-DNA were risk factors for HBV paternal transmission (OR=5.7,95％CI:1.1-29.1 ; OR=4.2,95％CI:1.7-10.0; OR=6.7,95％ CI:2.4-18.9).5)Dose-response relationships were seen between levels of paternal serum HBV-DNA load and cord blood HBV-DNA load,between levels of paternal serum HBV-DNA load and semen HBV-DNA load,between levels of semen HBV-DNA load and cord blood HBV-DNA load.6)Results from the analysis on ROC curve showed that paternal serum HBV-DNA load level (105 copies/ml) and semen HBV-DNA load level (103 copies/ml)were better demarcation points to forecast the occurrence of paternal transmission of HBV,because of the better sensitivity and specificity they had.Conclusion Items as positives on paternal serum HBV-DNA,paternal serum HBeAg and semen HBV-DNA were risk factors for HBV paternal transmission.When paternal serum HBV-DNA load ＞ 105 copies/ml and semen HBV-DNA load ＞ 103 copies/ml appeared,the positive rate of HBV paternal transmission would increase.

BACKGROUNDPericytes, located on microvessels, help to maintain vascular stability and blood-brain barrier integrity. The influence of pericytes on microvessels after spinal cord injury (SCI) is less clear. Therefore, the aim of this study was to investigate whether pericytes took a protective effect on microvessels in melatonin-treated SCI.

METHODSC57BL/6 mice were randomly divided into three groups: sham group, SCI group, and melatonin group (n = 27 per group). Functional recovery was evaluated using the Basso Mouse Scale. Motor neurons were observed using hematoxylin and eosin staining. Pericyte coverage was analyzed using immunofluorescence. Permeability of blood-spinal cord barrier (BSCB) was assessed by administration of Evan's Blue. Protein levels of occludin, aquaporin-4 (AQP4), angiopoietin-1 (Ang1), intercellular cell adhesion molecule-1 (ICAM-1), Bcl-2, and Bax were determined using Western blotting. Mimicking the pathological conditions of SCI, melatonin-treated primary pericytes were subjected to oxygen-glucose deprivation/reperfusion (OGD/R). Secretion of Ang1 was analyzed using an enzyme-linked immunosorbent assay, and the expression of ICAM-1 was detected by immunofluorescence.

RESULTSMelatonin treatment improved locomotor functional outcome and rescued motor neurons. Pericyte coverage was significantly reduced after SCI; melatonin treatment alleviated the loss of pericyte coverage and rescued perfused microvessels 7 days after injury. The permeability of BSCB and loss of occludin were attenuated, and edema formation and upregulation of AQP4 were inhibited, after melatonin treatment. The expression of Ang1 and Bcl-2 was improved, while the expression of ICAM-1 and Bax was inhibited, in melatonin-treated SCI mice. Furthermore, the secretion of Ang1 was increased and the expression of ICAM-1 was inhibited in melatonin-treated pericytes after OGD/R.

CONCLUSIONSMelatonin ameliorated the loss of blood vessels and disruption of BSCB to exert a protective effect on SCI, which might be mediated by increased pericyte coverage. The upregulation of Ang1 in pericytes could inhibit inflammation and apoptosis to protect the microvessels.

Angiopoietin-1 ; metabolism ; Animals ; Enzyme-Linked Immunosorbent Assay ; Intercellular Adhesion Molecule-1 ; metabolism ; Male ; Melatonin ; pharmacology ; therapeutic use ; Mice ; Mice, Inbred C57BL ; Microvessels ; cytology ; Occludin ; metabolism ; Pericytes ; drug effects ; metabolism ; Random Allocation ; Spinal Cord Injuries ; drug therapy ; metabolism