Objective:To screen the peptide binding to human bladder carcinoma cells specifically by using phage display technology in vivo.Methods: Nude mice were inoculated with bladder carcinoma cells BIU87 for establishing tumor-bearing mice model.The Ph.D.-C7CTM Peptide Library was injected intravenously via tail vein.Then we screened Phage containing exogenous peptides binding to bladder transitional carcinoma cells specifically.The phage peptide homed to the tumor tissues was obtained after 3 rounds screening in vivo.The phage clones affinity to BIU87 were identified by immunohistochemistry and ELISA.The positive peptide was synthetized by chemical methods after sequencing the positive monoclonal phage DNA.The tumor cell specificity of target peptide was identified by confocal laser scanning microscope and flow cytometry.Results:After 3 rounds screening in vivo,enrichment rate of phage was 4.334×102 times.Immunohistochemistry results showed that the dyeing of the tumor tissue had a rising trend following each round of phage screening,while liver had a lot of non-specific binding phage because the phages were metabolized through liver and kid-ney.The 30 phage clones were identified by ELISA and 10 clones had a strong affinity on BIU87 among 24 positive clones.Three amino acid sequences of positive phage clones were obtained.The highest rate of repeat sequences CSSPIGRHC(8/10) named NYZL1 and the FITC-C6-NYZL1 peptide was synthesized.Our results showed that it could bind to bladder carcinoma cells BIU87 specifically.Conclusion:We obtained the small molecular peptide NYZL1 binding to human bladder carcinoma specifically by means of phage display in vivo,which provide a theoretical basis for bladder carcinoma early diagnosis and targeted therapy.

Objective:New Zealand rabbits were immunized with VLPs-MEpS,VLPs-E2S,and the levels of neutralizing antibodies in serum were determined.Methods: New Zealand rabbits were immunized with 10 μg VLPs-MEpS and VLPs-E2S,serum was collected at diffferent time with a two-weeks interval.The neutralizing antibodies were determined by ELISA.HCV(type 1b) had been prepared and mixed with serum from immunized rabbit before infected Huh7.5 cell.The protection of neutralizing antibodies in serum was assessed.Results: Neutralizing antibodies had been induced in rabbit after immunized with VLPs-MEpS and VLPs-E2S.VLPs-MEpS group had higher titer of antibodies than that of VLPs-E2S group(P<0.05),both group had higher titer of antibodies than that of control groups significantly(P<0.01).VLPs-MEpS group had higher neutralization than that of VLPs-E2S group(P<0.05),the highest neutralization rate was 61.49%.Both groups were higher than control group notably(P<0.01).Conclusion: Protective neutralizing antibodies have been induced in New Zealand rabbit after immunized with VLPs-MEpS and VLPs-E2S.It′s the basement for development of neutralizing antibodies vaccine.

Objective: To investigate the effect of Shenmai Injection (Radix Ginseng Rubra, Radix Ophiopogonis) on angiogenesis and the expression of proliferating cell nuclear antigen (PCNA) in tumor tissue, and the mechanism of it in the treatment of tumor. Methods: The mouse tumor model was used to investigate the effect of Shenmai Injection on tumor growth on the whole. The expression of von Willebrand factor (vWF) and PCNA in tumor tissue was studied by means of immunohistochemistry. Results: Shenmai inhibited the tumor growth and reduced microvessel density and the expression of PCNA in tumor tissue. Conclusion:Antiangiogenesis is one of the mechanisms of Shenmai Injection in treatment of tumor.

Objective:To construct the recombinant prokaryotic plasmid to express HCV HLA-A2 restricted multi-CTL epitopes and to purify the fused protein for antigenic analysis.Methods:The human ubiquitin gene and multi-CTL epitopes gene was synthesized respectively,and digested by restrict enzyme before being cloned into pRSET-A.Then it was transformed into E.coli DH5α and the positive recombinant plasmid named pRSET-Ub-Mep was sequenced.Target protein was distinctly expressed after transformed into E.coli BL21 and induced with IPTG.Thus the protein was scanned and purified on Ni~(2+)-NTA column as well as Western blot performed after solubility analysis.Results:The recombinant plasmid pRSET-Ub-Mep was successfully constructed and it could efficiently express the target gene.Protein production was mainly in inclusion body and could be purified through Ni~(2+)-NTA column.The purified protein kept the antigen activity.Conclusion:The gene encoding for HCV HLA-A2-restricted multi-CTL epitopes is efficiently expressed and the target protein is purified,which establishes a foundation of further research to evaluate the cellular immune response induced by the target gene.

OBJECTIVETo examine the spatiotemporal profiles and localization of CysLT1R, CysLT2R and GPR17 in mice with 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced Parkinson disease (PD).

METHODSPD model was induced by subcutaneous injection of MPTP (25 mg/kg) for 5 d in adult male C57BL/6 mice. At d10 after MPTP injection, the expression and cellular localization of CysLT1R, CysLT2R and GPR17 in the substantia nigra were detected by immunohistochemistry and immunofluorescence.

RESULTSCysLT1R, CysLT22 and GPR17 were normally localized in TH-positive dopaminergic neurons and microglia, while CysLT2R was also expressed in astrocytes. In dopaminergic neurons, approximately 91% co-expressed GPR17, 77% co-expressed CysLT1R and 52% co-expressed CysLT2R. Compared with the control group, TH-positive cells in the substantia nigra were significantly reduced in PD mice. CysLT1R, CysLT2R and GPR17-positive cells were significantly reduced; and CysLT1R, CysLT2R, GPR17-positive dopaminergic neurons were also significantly reduced in the PD group. In the striatum, both CysLT1R and GPR17 were normally expressed in neurons; whereas CysLT2R was expressed in astrocytes. In PD striatum, CysLT1R and GPR17-positive cells were decreased, but CysLT2R expression was significantly increased which mainly expressed in the proliferating astrocytes.

CONCLUSIONCysLT1R, CysLT2R and GPR17 may be involved in the MPTP-induced PD damage in mice.

Animals ; Brain ; metabolism ; Disease Models, Animal ; Male ; Mice ; Mice, Inbred C57BL ; Nerve Tissue Proteins ; metabolism ; Parkinson Disease ; metabolism ; Receptors, G-Protein-Coupled ; metabolism ; Receptors, Leukotriene ; metabolism

OBJECTIVETo investigate the effects of CysLT receptor agonist leukotriene D4(LTD4) and antagonists on activation of microglia BV2 cells.

METHODSThe expression of CysLT1 and CysLT2 protein was determined by Western blotting and immunostaining in microglia BV2 cells. BV2 cells were pretreated with or without CysLT1 receptor selective antagonist montelukast, CysLT2 receptor selective antagonist HAMI 3379, or CysLT1/CysLT2 receptor dual antagonist BAY u9773 for 30 min, then the cells were treated with LTD4 for 24 h. Cell viability was detected by MTT reduction assay. Phagocytosis and mRNA expression of IL-6 were determined by fluorescent bead tracking and RT-PCR, respectively.

RESULTSIn BV2 cells, LTD4 did not affect proliferation but significantly enhanced phagocytosis and increased IL-6 mRNA expression in a concentration-dependent manner. LTD4 at 100 nmol/L induced a 1.4-fold increase of phagocytic index and a 2-fold up-regulation of IL-6 mRNA expression (P<0.01). HAMI 3379 and BAY u9773 (100 nmol/L) further increased LTD4-induced phagocytosis; BAY u9773 and montelukast decreased LTD4-induced IL-6 mRNA expression, while HAMI 3379 had no effect on that.

CONCLUSIONLTD4 activates BV2 cells in vitro and enhances IL-6 mRNA expression mediated by CysLT1 receptor, LTD4 induces phagocytosis which might be negatively regulated by CysLT2 receptor in BV2 cells.

Acetates ; pharmacology ; Cell Line ; Cell Proliferation ; Cyclohexanecarboxylic Acids ; pharmacology ; Humans ; Interleukin-6 ; metabolism ; Leukotriene Antagonists ; pharmacology ; Leukotriene D4 ; pharmacology ; Microglia ; cytology ; metabolism ; Phagocytosis ; Phthalic Acids ; pharmacology ; Quinolines ; pharmacology ; Receptors, Leukotriene ; metabolism ; SRS-A ; analogs & derivatives ; pharmacology

OBJECTIVE: : To determine the effects of cysteinyl leukotriene receptors (CysLTR and CysLTR) on phagocytosis of mouse BV2 microglial cells. METHODS: : BV2 cells were stimulated with microglial activators lipopolysaccharide (LPS) or CysLT receptor agonists LTD. The phagocytosis of BV2 cells was observed by immunofluorescence analysis and flow cytometry. The intracellular distributions of CysLTR and CysLTR in BV2 cells were examined with immunofluorescence staining. RESULTS: : Both LPS and LTD could significantly enhance the phagocytosis of BV2 cells, and such effect could be inhibited by CysLTR selective antagonist Montelukast and CysLTR selective antagonist HAMI 3379. The activation of BV2 cells induced by LTD or LPS resulted in changes in intracellular distributions of CysLTR and CysLTR. CysLTR and CysLTR was co-localization with a similar distribution. CONCLUSIONS : CysLTR and CysLTR regulate the phagocytosis of mouse BV2 microglial cells with a synergistic effect.
Acetates ; pharmacology ; Animals ; Cell Line ; Cyclohexanecarboxylic Acids ; pharmacology ; Lipopolysaccharides ; pharmacology ; Mice ; Microglia ; cytology ; Phagocytosis ; drug effects ; Phthalic Acids ; pharmacology ; Protein Binding ; drug effects ; Quinolines ; pharmacology ; Receptors, Leukotriene ; agonists ; metabolism