Exploration of Bloom's taxonomy in medical microbiology teaching / 中华医学教育探索杂志

In order to improve the teaching quality of medical microbiology, teachers constructed the syllabus from six levels according to Bloom's taxonomy, including memory, understanding, application, analysis, evaluation and innovation, teaching objectives, teaching methods and lesson preparation. The aim is to stimulate students' enthusiasm for learning and to promote students' ability of deep learning and innovation.

Purification,antiserum preparation and antigenic characters of ClpC2 in Myco-bacterium tuberculosis / 中国免疫学杂志

Objective:To investigate the antigenicity of ClpC 2 and the feasibility of polyclonal antibodies of ClpC 2 as detected antibody.Methods:rClpC2 was induced with IPTG.The rClpC2 was identified by SDS-PAGE and Western blot ,and purified by affinity chromatography ,with which rabbit were immunized and the specificity of rabbit antiserum was detected by Western blot , the titer of rabbit antiserum against ClpC2 was detected by double immunodiffusion and indirect enzyme-linked immunosorbent assay (ELISA).The antigenicity of the rClpC2 was detected by ELISA.The polyclonal antibodies of ClpC 2 were prepared to detect the ClpC 2 in clinical serum of TB patients by ELISA.Results:SDS-PAGE showed specific protein band with a relative molecular mass of 46 kD.The rClpC2 could bind with the antibody in the blood serum of the mouse immuned by MTB.By Bandscan analysis rClpC 2 accounted for about 58.7%of the total bacteria protein ,the purity of rClpC2 was 88.5% after purification.The ClpC2 of BCG could bind with the rabbit antiserum.The titer of antiserum were 1∶32 and 1∶320 000 by double immunodiffusion and ELISA detected respectively.ELISA results showed that clinical serum positive rate of rClpC 2 antigen was 46%in TB patients,the sensitivity of this protein was 46%,and the spe-cificity of this protein was 90%.ELISA results showed that the sensitivity of rabbit antiserum against ClpC 2 was 40%, and the specificity was 90%.Conclusion: Successfully expressed and purified rClpC 2 and high titer polyclonal antibody were successfully prepared,and these results will provide basements for further study on the biological functions of ClpC 2 and its candidate potentiality as serological diagnosis and drug-target and biological functions of antiserum against ClpC 2.

Effect of hsa-microRNA-218 on granulysin expression / 中国免疫学杂志

Objective:To elucidate the effect of hsa-microRNA-218(hsa-mir-218)on exogenous granulysin (GLS) expression in 293T cells.Methods:Total RNA was extracted from THP-1 cells induced by phorbol 12-myristate 13-acetatefor (PMA), and GLS gene was amplified by RT-PCR, and then cloned into pDsRed-Express-C1 to construct the GLS expression vector pDsRed-GLS.Then 293T cells were co-transfected with pDsRed-GLS and pGenesil-mir-218 (pGenesil-mir-control) and laser confocal microscopy was per-formed 36 h later to detect their co-expression .Total RNA and protein were extracted 48 h post transfection , and RT-PCR and Western blot were performed to detect the effect of hsa-mir-218 on exogenous GLS expression .Results:The GLS expression vector pDsRed-GLS was constructed successfully and laser confocal microscopy indicated that it was co -expressed with the interference vector .Compared with that of cells transfected with pGenesil-mir-control, Western blot showed a markedly decrease of GLS protein expression (50%) in the cells transfected with pGenesil-mir-218.However, GLS mRNA expression remained unchanged .Conclusion: hsa-mir-218 nega-tively regulates GLS expression at a post-transcriptional level , and this provides an experimental basis for future study of mechanism of GLS expression regulated by mir-218 .

Construction and practice of Pathogen biology quality courses / 中华医学教育探索杂志

Pathogen biology is a main course of curricula in medical university.In developing quality courses,we optimized Pathogen biology course content,created the PBL teaching mode suitable for this course,set up network teaching platform,enhanced teachers' qualification,constructed comprehensive and exploratory experiment teaching system,wrote new teaching syllabus and other measures,made the Pathogen biology course construction more reasonable and perfect.

Bactericidal activity of human granulysin expressed in macrophages against intracellular Mycobacterium tuberculosis / 中华传染病杂志

Objective To construct eukaryotic expression recombinant plasmid containing human granulysin(GLS) and investigate the effect of GLS expression in macrophage RAW264.7 cells on the bactericidal activity against intracellular Mycobacterium tuberculosis.Methods GLS gene was amplified by nested-polymerase chain reaction(PCR) from human cytotoxicity T lymphocyte(CTL) activated by allogenic antigen,and inserted into pBudCE4.1 vector to construct recombinant plasmid.Subsequently,the plasmid was transfccted into RAW264.7 cells which were infected with Mycobacterium tuberculosis H37Rv.The expression of GLS was detected by nested-PCR and immunocytochemistry method.The RAW264.7 cells were lysed after transfected for 96 h,then acidfast stained,cultivated and colony count were done to determine the intraeellular bactericidal activity of GLS.The data were analyzed by t or t' test.Results The pBudCE4.1/GLS eukaryotic expression recombinant plasmid was successfully constructed.The transcriptional and translational expressions of target gene GLS were detected in RAW264.7 cells which were infected with Mycobacterium tuberculosis H37Rv.The bacterial load in macrophages of phorbol myristate acetate(PMA)+pBudCE4.1/GLS group,PMA+pBudCEA.1 group and non-activated group were 1.44±1.25,3.16±0.20 and 3.59±0.21,respectively.The differences between groups were all significant (t=2.403,t=2.854,both P＜0.05).Conclusion Eukaryotic expression recombinant plasmid carrying human GLS gene expressed in macrophages has strong bactericidal activity against intracellular mycobacteria,which provide information for the further study on therapeutic vaccine against Mycobacterium tuberculosis.

Study of new idea of subject integration of pathobiology / 中华医学教育探索杂志

This article aims at exploring ways of integrating subject of pathobiology under the guidance of scientific outlook on development and improving and enriching pathobiology inte-gration and construction through curriculum systems recombination and integration,teaching con-tents optimization to reflect the latest research progress,network education platform establish-ment,comprehensive and exploratory laboratory course teaching system construction,new syl-labus and teaching material making up.

The impact of 5-Aza-CdR on demathylation of transcription regulation of E-cadherin gene in tumor cell line / 中国癌症杂志

Background and purpose:To explore the possibility of genetic re-expression silenced by DNA aberrant hypermethylation which is a common epigenetic modification in carcinogenesis. 5-Aza-CdR, an inhibitor of DNA methylation, was used to determine the effects of expression of tumor suppressor gene E-cadherin in tumor cell lines. Methods:Methylation specific PCR(MSP) was utilized to examine methylation status of E-cad gene on breast carcinoma cell line MDA-MB-435 before and after the treatment with 5-Aza-CdR. Immunohistochemistry(IHC) was used to test the expression of E-cad protein. Semi-quantitative RT-PCR method was used to detect the changes of E-cad mRNA.Results:1).E-cad methylation was positive(116bp) and unmethylation was negative on MDA-MB-435 cell before the treatment with 5-Aza-CdR. After being treated with 5.0umol/L 5-Aza-CdR for 3 days, methylation turned negative and unmethylation positive bands(97bp) were detected. 2).The E-cad protein expression was not detected by immunohistochemistry on MDA-MB-435 cell before the treatment, while E-cad staining was positive on the cell membrane after the treatment. 3). The E-cad mRNA failed to be amplified in cells before the treatment. After incubation at variable concentrations of 0.5 ?mol/L, 1.0 ?mol/L, 2.0 ?mol/L and 5.0 ?mol/L 5-Aza-CdR for 3 days, respectively, E-cad mRNA expression was detected on the fourth day in a dose-dependent manner. Correlation between the mRNA expression level and the agent concentration was observed.Conclusions:The demethylation agent 5-Aza-CdR can reverse the aberrant E-cad methylation status in MDA-MB-435 and re-expressed E-cad mRNA and protein.

Reforming of optimizing microbiology curriculum systems to different specialties / 中华医学教育探索杂志

To fully incarnate"specialization"peculiarities of microbiology teaching courses by means of optimizing microbiology teaching contents and curriculum systems,and making up a new syllabus suitable to different specialties will lay a very solid foundation for cultivating medical talents with specialty traits.

Eukaryotic expression and identification of recombinant shuttle plasmid for co-expression of human granulysin active peptide and murine IL-12 / 第三军医大学学报

Objective To construct an eukaryotic co-expression plasmid pBM9 carrying human granulysin active peptide and murine IL-12 and determine its expression.Methods The primer pairs including granulysin leader peptide DNA sequence were designed to amplify granulysin from the plasmid pZM03 carrying granulysin gene by polymerase chain reaction(PCR).PCR product was directly cloned into an eukaryotic co-expression plasmid pBudCE4.1 to construct plasmid pBudCE4.1-S9K.pBudCE4.1-S9K plasmid was identified by DNA sequencing.Murine IL-12 gene was subcloned into pBudCE4.1-S9K to construct eukaryotic co-expression plasmid pBudCE4.1-S9K/mIL-12.Mycobacteria replicon Orim from plasmid pZM03 was subcloned into NotⅠsite of pBudCE4.1-S9K/mIL-12 to construct eukaryotic co-expression shuttle plasmid pBM9.pBM9 was tansfected into RAW264.7 cells.RT-PCR was used to detect the mRNA expressions of granulysin and IL-12.The protein expression of them were observed by immunocytochemical method and ELISA respectively.Results RT-PCR identified the expressions of granulysin and mIL-12 in transfected cells and culture supernatant.Immunocytochemical method and ELISA verified the protein expressions.Conclusion The recombinant shuttle plasmid pBM9 is successfully constructed and expressed in vitro,which laid a foundation of gene therapy for tumors with granulysin and mIL-12.

The cDNA cloning of human granulysin Mr 15000 and Mr 9000 active segments from CTL activated by allogenic antigen / 重庆医科大学学报

Objective; To clone, sequence and analyze the coding sequences of the Mr 15000 and Mr 9000 active segments of natural granulysin derived from human CTLs activated by allogenic antigen ,in order to establish the basis for further purifying and investigating the immune - impairing mechanism of granulysin. Methods; The coding sequences of the Mr 15000 and Mr 9000 granulysin gene were amplified from the total RNA of activated CTLs of healthy person after reverse transcription by Nested - PCR, inserted into pET32a ( + ) vectors and then transformed into E. Coli TOP10, respectively. The recons were identified by PCR, endonuclease digestion and sequencing. Results: The whole coding sequences of the Mr 15000 and Mr 9000 active segments were successfully cloned as expected. The accurate pET32a - Mr 15000 and pET32a - Mr 9000 recons were obtained through Colony - PCR, endonuclease digestion and sequencing. There lied polymorphism on the 119 th amino acid of the product encoded by GLS gene. Conclusion; The coding sequences of the Mr 15000 and Mr 9000 active segments of human granulysin can be obtained and cloned by the methods mentioned above and can be used for subsequent research.

Effect of granulysin (GLS) and IL-12 genes' expression products on proliferation and apoptosis of melanoma B16 cell / 重庆医科大学学报

Objective:To dectect the effect of granulysin and IL-12 genes'expression products on proliferation and apoptosis of melanoma B16 cell in vitro.Methods:Co-expression plasmid including granulysin peptide and murine interleukin 12(mIL-12)genes was transfected into melanoma B16 cell with Lipofectamin TM2000 and its expression products were detected by RT-PCR.Growth suppression was detected with MTT colorimetric assay,and cell apoptotic alterations were evaluated by Hoechst 33258 staining,AO/EB staining,and Annexin V-FITC flow cytometry(FCM).Results:GLS peptite and IL-12 genes could be expressed in B16 cells.Expression products inhibited the proliferation of melanoma cells under MTT observaton.Cells apoptosis with nuclear chromatin condensation,fragmentation and cell membrane change were observed under Hoechst 33258 staining and AO/EB staining.FCM analysis showed the apoptotic rates in test group was 21.02%,which was higher than that in control in control group(15.57%).Conclusion:Expression products of granulysin and mIL-12 genes can not only inhibit proliferation but also induce apoptosis of murine melanoma cell line B16 in vitro.