Objective To investigate the inhibition of pathogenic viral gene expression in HPV6bE6-positive cell line by specific siRNA, which might have great potential for clinical use. Methods B16 cells were transfected with recombinant plasmid pcDNA3.1(+)-GFP/HPV6bE6, and the positive cell clones were selected by fluorescence protein observation and RT-PCR. Four specific siRNAs, none of which shares homology with exons of known human genes, were designed and synthesized to target HPV6bE6 mRNA. Quantitative real-time PCR was performed to measure the inhibition rates of target gene expression by comparing HPV6bE6 mRNA concentrations before siRNA transfection with those after transfection. The inhibition rates of target gene expression with different siRNA concentrations of 0.2 nmol/L, 1 nmol/L, 10 nmol/L, 50 nmol/L, 150 nmol/L and with different treatment time at 24 h, 48 h, 72 h and 96 h after transfection were measured respectively. Results More than 90% reduction of HPV6bE6 mRNA was observed following treatment with HPV6bE6-siRNA, and HPV-negative cells were apparently unaffected by HPV6bE6-siRNA. The decrease of HPV6bE6 mRNA was maximal at 24 h after siRNA treatment and sustained for at least 4 days. The minimal level of siRNA to efficiently silence the homogeneous target gene HPV6bE6 was 1 nmol/L. Conclusion HPV6bE6-siRNA can efficiently and specifically silence target genes and may be developed as a potential therapeutic approach for HPV infection.

Objective To investigate the cellular immune response to the recombinant DNA vaccine CRT120/HPV6bE7 in mice. Methods The recombinant encoding HPV6bE7, linked with CRT120, was constructed in pcDNA3.1 eukaryotic vector with the report gene GFP. The pcDNA3.1-GFP -HPV6bE7 was transfected into B16 cells by a lipofectamine kit. The C57BL/6 mice were vaccinated with recombinant DNA plamids. The T-cell phenotype in the peripheral blood lymphocytes of the immunized mice was measured by flow cytometry. The CTL activity of the lymphocytes from the spleens and lymph nodes, and the levels of IL-2 and IFN-? were analyzed. Results The constructed recombinant plasmid CRT120/HPV6bE7 was analyzed. Positive transfected cell clones were established and could stably express the target gene HPV6bE7. Compared with HPV6bE7, CRT120/HPV6bE7 plasmid enhanced to a greater extent CD8+ T-lymphocyte differentiation, the number of TCR?? T-lymphocytes and the levels of IL-2 and IFN-? (all P

Objective To investigate the effects of crude extracted proteins from lesions of condyloma acuminata on immunophenotypes of dendritic cells.Methods Plastic-adherent mononuclear cells(MNCs)were isolated from umbilical cord blood or peripheral blood and cultured in media containing cytokines(GM-CSF,IL-4and LPS).The morphology and phenotypes of these cells were analyzed by flow cytometry and microscopy in12day's culture.Cells on the fourth day were incubated with crude extracts from lesions of condyloma acuminata,foreskin proteins,and PBS,respectively,followed by phenotypic analysis after9-12days' culture.Results Expression of antigens CD1a,CD80,CD86,MHC-I,MHC-II,CD14,CD54was detected after12days' cul-ture.It was shown that MNCs could be induced to differentiate to mature dendritic cells in our culture system.After incubation with crude extracts from lesions of condyloma acuminata for another9-12days,expression of CD86and HLA-DR was increased on dendritic cells.Conclusions Compared to foreskin and PBS pulsed dendritic cells,expression of CD86and HLA-DR is upregulated on dentritic cells after pulsing with condyloma acuminata lesion proteins.The data suggest that crude extracts from lesions of condyloma acuminata might enhance antigen-pre-senting capacity of dendritic cells and strengthen activation of T lymphocytes.

Objective To construct four expression plasmids, pcDNA3.1-GFP/HPV6bE6, pcDNA3.1-GFP/HPV6bE7, pcDNA3.1-GFP/HPV11E6, pcDNA3.1-GFP/HPV11E7 and their transfected murine cell lines. Methods The Four recombinant expression plasmids comprising HPV6bE6,HPV6bE7,HPV11E6 and HPV11E7 linked with GFP, respectively, were constructed and transfected to B16 cells by lipofectamine kit. Positive clones were selected by G418 and observed by fluorescent microscopy and identified by RT-PCR. Results The four constructed recombinant plasmids were authenticated by restriction enzyme digestion and DNA sequencing. Under the fluorescent microscope, the green fluorescence could be observed in cytoplasm and nucleus of four transfected B16 cell lines. The RNA extracted from positively transfected clones resistant to G418 were analyzed by RT-PCR, which demonstrated the presence of four expected fragments. Conclusions The transfected murine cell lines B16 can express HPV6bE6,HPV6bE7,HPV11E6 and HPV11E7 gene. These transfected cell lines can be further transplanted to mice in order to investigate the biological properties and immunological mechanisms of these genes in vivo.

Objective To screen and identify the predicted epitopes of synthesized HLA-A*0201restricted CTL derived from HPVll E7 antigen.Methods Five HPVll E7 CTL epitope peptides and terramers consisting of HLA-A*0201 were selected by way of computer and synthesized by Sanquin company,including HPVllE7 7-15(TLKDIVLDL),15-23(LQPPDPVGL),47-55(PLTQHYQIL),81-89(DLLLGTLNI)and 82-90(LLLGTLNIV).These peptides binding to human peripheral blood-derived DCs were tested for their ability to activate T cells isolated from peripheral blood lymphocytes of HLA-A*0201 healthy individuals.the number of specific tetramer+CD8+T cells by flow cytometry,the level of the section of IFN-γ by ELISA,and the ability of the CTL to kill the target cells were observed.Results The immature DCs could be fully activated by all the five HPV11 E7 peptides.Peptide-loaded mature DCs were able to stimulate the epitope-specific T cells responses in vitro.An increased frequency(P<0.05)of T ceils specific for the E7 7-15 epitope compared to other epitopes of HPV11E7.The epitope-specific CTL of E7 7-15 induced by the activated DCs specifically killed HPV11E7 expressing 293 cell line,and in a ratio of 50:1,the specific cytolytic activity was the strongest than the others(P<0.05).Conclusion DCs loaded with HPV11 E7 7-15(TLKDIVLDL)peptide can induce highly effective and specific ectogenic processed epitopespecific CTL responses in vitro.This peptide may be the candidate for development of CTL based vaccine in the treatment of HPV infeetions.

Objective To investigate the epidemic factors of human brucellosis in Qiqihar from 2008 to 2014 so as to provide a scientific basis for prevention and control of the disease.Methods Descriptive epidemiologic method was used to analyze the monitoring results of human brucellosis in Qiqihar between 2008 and 2014.Indexes observed were:the incidence rate,the distribution of gender,age,occupation,area and time.Results From 2008 to 2014,there were a total of 15 003 cases of brucellosis covering all counties.The average incidence rate was 38.85/100 000.The average incidence rate of Meilisi Daur District was the highest,178.93/100 000.Cases occurred year around and peaked in March-July.The average incidence rate of male and female brucellosis was 57.53/100 000 and 19.35/100 000,respectively,and male patients were more than female (x2 =3 658.973,P ＜ 0.05).Most patients were in the 41-50 years old age group,and the number of patients was 4 386.The major occupation was farmer (herdsmen),which acount for 93.96％ (14 097/15 003).Conclusions Qiqihar is the epidemic area of brucellosis and has a higher incidence.Governments should pay nore attention to the epidemic of brucellosis.Health education and intervention measure for high-risk population should be strengthened and the professional team should be reinforced so as to reduce the incidence of brucellosis.

OBJECTIVE: To investigate the ING1 gene mutation status in human laryngeal squamous cell carcinoma(LSCC), and the association of p33(ING1b) protein expression with p53 protein expression. METHOD: DNA of LSCC tissue was extracted, and nucleotide of the second exon was amplified and sequenced to determine the chromosome status. The p23(ING1b) and p53 protein expression were detected by immunohistochemistry and the association between them were analyzed. RESULT: No mutation was detected in ING1 gene, but a single polymorphism from GGG to AGG at codon 170 of ING1 gene was found in 2 of the 25 LSCC tissues. The immunohistochemical analysis showed that 4 had positive p33(ING1b) expression. No association was found between p33(ING1b) expression and LSCC clinical features, or between p53 and clinical features. However, significant difference was found between p33(ING1b) and p53 expression. p33(ING1b) tended to be negative in p53 expression positive tissue. CONCLUSION ING1 gene mutation appears rare in LSCC. In normal physical condition, p33(ING1b) may play a synergistic effect with p53 protein.
Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Female ; Genes, Regulator ; Humans ; Inhibitor of Growth Protein 1 ; Intracellular Signaling Peptides and Proteins ; genetics ; Laryngeal Neoplasms ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; Mutation ; Nuclear Proteins ; genetics ; Tumor Suppressor Protein p53 ; metabolism ; Tumor Suppressor Proteins ; genetics