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Objective To explore the effects of anti-tumor immunity induced by dendritic cells (DCs) vaccine pulsed with PEP3-KLH (keyhole limpet hemocyanin) on induction of specific immunity against prostate cancers.Methods DCs were propagated from bone marrow of C57BL/6 mice in vitro with granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4).On day 5 of culture, DCs were harvested and incubated with PEP3-KLH or KLH.Then the DC vaccine was inoculated into C57BL/6 mice by subcutaneous injection for three times with an interval of two weeks.One week after the last vaccination, the levels of IL-2, IL-12, and interferon (IFN)-γwere measured with enzyme-linked immunosorbent assay (ELISA) kit.The aforementioned immunized mice with DC vaccines were challenged with transgenic adenocarcinoma of mouse prostate (TRAMP)-C2 tumor cells, the tumor growth curve was drawn, and survival rate was checked and compared.The cytotoxic T lymphocyte (CTL) activity induced by DCs was tested with lactate dehydrogenase (LDH) release method.The percentages of CD3 + , CD4+ ,or CD8 + T cells in tumor-infiltrating lymphocytes (TILs) were determined with flow cytometry.Results PEP3-KLH-DC group stimulated the body and induced higher levels of secreted IL-2, IL-12, and IFN-γ compared to DCs control and KLH-DC groups (P ＜ 0.01).The tumor of mice vaccinated with PEP3-KLHDC grew significantly slower than that injected with DCs or KLH-DC (P ＜0.01).Compared to the others, the survival rate in PEP3-KLH-DC group raised remarkably (P ＜ 0.01).PEP3-KLH-DC group induced more outstanding specific CTL activities of killing tumor cells than DCs control group and KLH-DC group (P ＜ 0.01), and the cytotoxicities of TILs in PEP3-KLH-DC group was significantly enhanced (P ＜0.01).The percentages of CD3 + , CD4 + , or CD8 + T cells in TILs (40.9％, 34.1％) in PEP3-KLH-DC group were significantly higher than those in DC-KLH (27.3％, 5.2％) or DCs (26.2％, 5.1％) group.Conclusions PEP3-KLH-DC vaccine can inhibit effectively tumor growth, enhance long-term survival in mice,intensify the local immunologic function of tumor, and elicit and promote profound specific anti-prostate cancer cellular immune responses.
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Proteomics is an emerging discipline and has been widely used in a variety of fields despite of having very short history in comparison with other disciplines. In Chongqing Medical Univer-sity, the course contents were adjusted to fulfill the most effective integration of proteomics research with postgraduate training program for medical university. Diverse teaching was advocated here and af-ter-school communications were greatly encouraged in teaching. Traditional multimedia teaching plat-form remained the main teaching way and students were organized to visit the research platform as supplementing teaching way. The overall quality and effectiveness of teaching were effectively improved by successful implementation of the above initiatives.
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Objective:To express recombinant protein mIL-21-hIgGFc in 293E cells,and investigate its effect on CD8+T cell.Methods:Total RNA was extracted from the mouse spleen cells ,and then IL-21 gene was amplified by RT-PCR and inserted into expression vector PTT3-hIgGFc.PTT3-mIL-21-hIgGFc were transfected into 293E cells by calcium phosphate method.The supernatants were collected at 48 hours and 72 hours and concentrated by MOLLIPORE Labscale TM TFF system ( 5 kD membrane ).The mIL-21-hIgGFc fusion protein was purified with HiTrap TM Protein G column.The protein was quantified by SDS-PAGE and ELISA.The biological activity of the protein was determined by detecting the change of the phenotypes of CD 8+ T cells treated with the protein.Results: The constructed recombinant plasmid PTT 3-mIL-21-Fc was confirmed by sequencing.PTT3-mIL-21-Fc was transfected into 293E cells,mIL-21-Fc protein in culture supernatant was collected after 48 hours and 72 hours.The protein in cell su-pernatant reached a concentration of 787 ng/ml which was determined by ELISA.The protein was purified by Protein G chromatography column.P1A-specific T cells were treated with mIL-21-hIgGFc, and found that the CD44low CD62Lhi CD8+ population increased compared to the control.Conclusion:We built PTT3-mIL-21-hFc recombinant plasmid, expressed mIL21-hFc fusion protein in 293E cells,and purified by Protein G column.By treating mIL-21-hFc ,the antigen-primed CD8+T cells prefer to differentiate into CD44low CD62Lhi CD8+T cells which had been reported as a memory stem phenotype .This protein may be used to improve the effectness of adoptive T cell cancer therapy.
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In this paper,the recent advances in both biomedical sciences and higher medical education reform were reviewed and analyzed.Furthermore,we proposed and reconstructed the teaching system of biochemistry and molecular biology course in our university,including its teaching content,teaching methods,teacher team,teaching management,etc.The preliminary practice of this system has obtained significant positive effects on teaching quality and student performance.
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In order to strengthen the graduate management in scientific research platform and to ensure the quality of graduate training,the ideological and moral education was invigorated through establishing virtual party branch,the behavior was regulated through establishing and amplifying the daily management system,the student interests were protected through establishing financial management system and the cultivation quality was guaranteed through perfecting the academic management system.Satisfactory results were achieved in molecular medicine and cancer research center in Chongqing Medical University.
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Molecular medicine and cancer research center in Chongqing Medical University adhered to academic honesty and credit education ,established original laboratory records system , regularly carried out seminar and improved paper submission program thus to reject academic miscon-ducts from the source,guarantee the authenticity of the data and improve the academic moral level of postgraduates.
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X-box binding protein1 (XBP1) is an important transcription factor, which participates in many signal transduction procession.To investigate the biological function of XBP1, yeast two-hybrid system to screen proteins interacting with XBP1 in hepatocytes was performed. The XBP1 coding sequence was amplified by polymerase chain reaction (PCR) method, and was cloned in pGEM-T vector. After the target region was sequenced, it was subcloned into the bait plasmid pGBKT7, then was transformed into yeast AH109(a type). After the expression of bait plasmid pGBKT7-XBP1 in AH109 yeast strains were proved by Western blot. The transformed yeast AH109 was mated with yeast Y187(α type) containing hepatocyte cDNA library plasmids pACT2 in 2 ×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medilium (SD/-Trp-Leu-His-Ade) containing X-α-gal for selection and screening. After sequencing and verifying ORF of positive colonies,7 different kinds of proteins were obtained. In order to further testify the interaction between the screened proteins and XBP1, one of positive colonies MT1E was cloned. The interaction between MT1E and XBP1 in vitro/in vivo were examined successfully with GST pulldown and coimmunoprecipitations. It was shown that MT1E would be a new regulatory protein of XBP1. These screened proteins by yeast two-hybird system were closely correlated with liver fundamental metabolism,protein synthesis and transport,cell proliferation and apoptosis. The results mentioned above contributed to reveal the XBP 1 biological function, and brought some new clues for further exploration of the expressing and regulating mechanism of XBP1.
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Objective To learn the effect on carcinoma xenograft in nude mice by inhibiting human papillomavirus 16(HPV16) E6 gene expression in CaSki cell. Methods The recombinant plasmids expressing HPV16 E6 small interference RNA (siRNA) were transfected into CaSki cell. The cells expressing recombinant plasmid was screened out with G418. The expression of E6 mRNA was determined by RT-PCR. The cells were inoculated into BALB/c nude mice subcutaneouly and the growth of the xenograft carcinoma was observed. After the pGensil-CH2 recombinant was injected into the carcinoma, the growth of carcinoma and pathological changes of carcinoma were observed. Results The CaSki cell expressing E6 siRNA was obtained, and HPV16 E6 mRNA expression in CaSki cell was down-regulated. The oncogenicity of the CaSki cell expressing E6 siRNA was degraded, the inhibition rate was up to 71.4% as compared with that of control group. The growth of tumor in nude mice was inhibited after the E6 siRNA plasmids were injected into the nude mice. The volume and weight of the tumor treated by siRNA were smaller than that of control group significantly. More necrotic area and less cell division phase were observed under light microscope in the E6 siRNA treated tumor. Conclusion The oncogenicity of the CaSki cell was degraded after silencing HPV16 E6 gene in CaSki cell by E6 siRNA.
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Aim To construct a eukaryotic expression vector of human MCHR2 and to analyze the stable expression and signal transduction pathways of MCHR2 in CHO cells. Methods The full-length MCHR2 cDNA fragment was amplified by PCR from the human fetal brain cDNA library and inserted into eukaryotic expres-sion vector pcDNA3.1(+), resulted in the recombinant expression vector pcDNA3.1-MCHR2. The recombinant plasmid was confirmed by the restriction enzymatic digestion and DNA sequencing analysis, and then transfected into CHO cells by lipofectamine. A stably-transfected cell line was obtained by the dominant G418 selection, and the expression of the MCHR2 gene on transcription and translation levels were identified by RT-PCR and Western blot. Signal transduction pathways mediated by MCHR2 were analyzed by measurement of intracellular cAMP and calcium. Results The eukaryotic expression vector pcDNA3.1-MCHR2 was successfully constructed and the MCHR2 gene was stably transfected into CHO cells. A stably-transfected cell line was established and the MCHR2 gene was efficiently transcribed and translated. MCH stimulation had no effect on the production of cAMP, however, it could induce a clear and transient increase of intracellular calcium, suggesting that MCHR2 was only coupled to Gq protein. Conclusion The stable expression of MCHR2 and analysis of its signal transduction pathways in CHO cell line provided a solid experimental foundation for further studies on the function of the MCHR2 gene in vitro.
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Objective To investigate the protein change in immature dendritic cells(DC) exposed to sendai virus replication sequence.Methods The total cellular proteins expressed in DC infected with Sev/△F or SeV were collected and quantitated,then separated by two-dimensional gel electrophoresis and visualized by silver staining.Digital images were analyzed by PDQuest software.The differentially expressed protein spots were picked,digested in gel and subjected to peptide mass fingerprinting using MALDI-TOF MS.Results Protein spots in 2-DE gel for DC treated by Sev/△F were more than that by SeV,among which a lot of proteins increased.Conclusion It was relevant to the replication sequence that proteins decreased in dendritic cells infected with sendai virus vector.