医学免疫学是各医学类专业本科生必修的基础医学课程,是连接基础医学和临床医学的桥梁学科.因此,学好医学免疫学对医学生尤为重要.医学免疫学实验教学不仅能加深学生对理论知识的理解,掌握基本的免疫学实验技能,还能培养学生的实践能力、探索精神、科学思维和创新能力.广州医科大学病原生物学与免疫学教研室对医学专业免疫学实验教学内容、方法和考核方式等进行了改革,不仅调动了学生实验的积极性,也提高了学生的实践能力与创新思维,达到了更好的教学效果.

Natural killer (NK) cells are classic innate immune cells that play roles in many types of infectious diseases. NK cells possess many kinds of TLRs that allow them to sense and respond to invading pathogens. Our previous study found that NK cells could modulate the immune response induced by Schistosoma japonicum (S. japonicum) in C57BL/6 mice. In the present study, the role of TLRs in the progress of S. japonicum infection was investigated. Results showed that the expression of TLR3 on NK cells increased significantly after S. japonicum infection by using RT-PCR and FACS (P < 0.05). TLR3 agonist (Poly I:C) increased IFN-γ and IL-4 levels in the supernatant of cultured splenocytes and induced a higher percentage of IFN-γ- and IL-4-secreting NK cells from infected mouse splenocytes (P < 0.05). Not only the percentages of MHC II-, CD69-, and NKG2A/C/E-expressing cells but also the percentages of IL-4-, IL-5-, and IL-17-producing cells in TLR3+ NK cells increased significantly after infection (P < 0.05). Moreover, the expression of NKG2A/C/E, NKG2D, MHC II, and CD69 on the surface of splenic NK cells was changed in S. japonicum-infected TLR3−/− (TLR3 KO mice, P < 0.05); the abilities of NK cells in IL-4, IL-5, and IL-17 secretion were decreased too (P < 0.05). These results indicate that TLR3 is the primary molecule which modulates the activation and function of NK cells during the course of S. japonicum infection in C57BL/6 mice.

Objective To explore the content of different subset of regulatory B cells in the spleen of Schistosoma japonicum infected C57BL/6 mouse. Methods C57BL/6 mice were percutaneously infected with Schistosoma japonicum(S. japonicum) cercariae. Five weeks later ,splenocytes were isolated ,and single cell solutions were made. The content of CD19 + B cells,and the expression of Immunomodulatory related molecule:CD5,CD11b,and PD-1 on B cells were investigated by the means of cell surface staining,and detected by Fluorescence activated cell sorting (FACs). Results After S. japonicum infection,the percentages and numbers of B cells in the spleen were increased significantly(P<0.01). The percentages of CD5+and CD11b+B cells were also increased(P<0.05). However,the changes of PD-1+B cells was not so clear (P>0.05). Conclusion S. japonicum infection could up regulate CD5+ and CD11b+ regulatory B cells in the spleen of C57BL/6 mice.

Objective To study the percentage of T lymphocyte and its subsets in follicular fluid of infertile women with and without polycystic ovary syndrome(PCOS). Methods Twenty infertile women diagnosed as PCOS(PCOS group) according to the Rotterdam criteria were enrolled in the study. The control group contains thirty-six normally ovulating infertile women(NOW group). The follicular fluid(FF) of each patient was collected and lymphocyte was enriched ,and then the percentage of CD3+ T,CD4+ Th,CD8+ Tc,and CD4+CD25+Treg cells was detected by flow cytometry. Results The percentage of FF CD3+T cells in patients with PCOS was significantly decreased compared with patients in NOW group [(66.2 ± 4.5)% vs. (54.8 ± 5.3)%,P<0.05]. In all three T lymphocyte subsets that were detected ,the percentage of FF CD8+ Tc cells was significantly lower in PCOS group than in NOW group[(28.4±3.5)% vs.(16.8±6.2)%,P<0.01],but no obvious change was found in the proportion of CD4 + Th or CD4 +CD25 +Treg cells between two groups (P>0.05). Conclusion The percentages of FF CD3+T and CD8+Tc cells were significantly lower in patients with PCOS than those in NOW patients,indicated an immune-dysfunction in PCOS patients.

Objective This study focuses on the effects of Toll?like receptor 2(TLR2)ligands on the expression of chemokines from THP?1 cells. Methods After treatment with PGN or LTA of different concentrations ,THP?1 cells were collected ,and mRNA was extracted from the collected THP?1 cells,then the extracted mRNA was subjected to quantitative real time PCR detection. The supernatant was subjected to ELISA detection. In inhibition experiment ,THP?1 cells were pretreated with different signaling molecule inhibitors ,then stimulated with PGN or LTA ,and finally detected by qPCR. Results PGN and LTA promoted mRNA expression of chemokines CCL2,CXCL8 and CXCL10 from THP?1 cells,and signaling molecule inhibitors differentially suppressed the mRNA expression of these chemokines. Conclusion The mRNA expression of chemokines CCL2,CXCL8 and CXCL10 from THP?1 cells by the stimulation of TLR2 ligands PGN and LTA was differentially regulated by the MAPK and NF?κB pathways.

Objective To investigate the molecular evolution characteristics of human coronavirus (HCoV) subtypes in patients with fever and respiratory tract infection in Guangzhou from 2010 to 2012.Methods Partial fragments of NP,RdRp and S genes of HCoV-OC43,HCoV-229E and HCoV-NL63 positive samples were amplified by RT-PCR and sequencing.Bioinformatics software,including Bio-edit,Mega4.0 and Clustal1.83 were used for comparison and analysis of NP,RDRp and S gene sequences.Molecular evolutionary tree of different gene regions of HCoV-OC43,HCoV-229E and HCoV-NL63 were built.Results No remarkable variation or recombinant strain of HCoV-OC43,HCoV-229E and HCoV-NL63 was found in Guangzhou during 2010-2012.The HCoV-OC43 substrains were genetically closest to the strains found in Belgium and Hong Kong (GenBank accession number JN129834 and AY903460).HCoV-229E substrains were genetically closest to those found in Amsterdam (GenBank accession number JX503060) and HCoV-NL63 most genetically close to those in Amsterdam and Beijing (GenBank accession number JX104161 and DQ445911).The NP and RDRp genes of all subtypes were highly conserved,while S gene was more variable.Conclusions There were at least 3 substrains of HCoV-OC43,HCoV-229E and HCoV-NL63 epidemic in Guangzhou during 2010-2012,and no remarkable variation or recombinant viral strain was found.The NP and RDRp genes of all subtypes were highly conserved and can be used in virus detection,while S gene was more variable and suitable for phylogenetic and variation study.

Natural killer cells (NK cells) and natural killer T cells (NKT cells) play a role in anti-infection, anti-tumor, transplantation immunity, and autoimmune regulation. However, the role of NK and NKT cells during Schistosoma japonicum (S. japonicum) infection has not been widely reported, especially regarding lung infections. The aim of this study was to research the NK and NKT cell response to S. japonicum infection in the lungs of mice. Using immunofluorescent histological analysis, NK and NKT cells were found near pulmonary granulomas. Moreover, flow cytometry revealed that the percentage and number of pulmonic NK cells in S. japonicum-infected mice were significantly increased (P < 0.05). However, the percentage and cell number of NKT cells were decreased compared to those of normal mice (P < 0.05). The expression of CD69 on pulmonic NK and NKT cells was increased after infection (P < 0.05), and CD25 expression increased only on NKT cells (P < 0.05). Intracellular cytokine staining showed a higher percentage of IFN-γ+ and lower percentage of IL-5+ pulmonic NK cells (P < 0.05) compared to controls. However, the percentage of IL-17+, IL-10+, and IL-5+ pulmonic NKT cells significantly increased (P < 0.05). Additionally, there was a significant decrease in NKG2A/C/E (CD94) expression and an increase of NKG2D (CD314) expression on pulmonic NKT cells (P < 0.05), which might serve as a mechanism for NKT cell activation during S. japonicum infection.

Objective:Immune cells and inflammatory mediators play important roles in the development of atherosclerotic vascular inflammation.This study focuses on the direct effect ofIL-27 on human coronary artery endothelial cells.Methods:In this study,the effects of IL-27 and TNF-α on inflammatory cytokines and chemokines were investigated.Results:IL-27 could significantly enhance the TNF-α-mediated upregulation of inflammatory cytokine IL-6 and chemokines CCL5 from human coronary artery endothelial cells.The release of IL-6 and CCL5 was significantly suppressed by specific signaling molecule inhibitors.Conclusion:The induction of these mediators from the human coronary artery endothelial cells could be differentially regulated by the c-Jun N-terminal kinase,p38 mitogen-activated protein kinase,and nuclear factor-κB pathways.These results provided new insights into the effect of IL-27 on the TNF-α mediated activation of human coronary artery endothelial cells in atherosclerotic vascular inflammation.

医学微生物学实验教学的目的在于使学生掌握微生物学基本实验技术,并通过实验操作更好地理解理论知识,培养学生分析与解决问题的能力.教研室对临床医学专业医学微生物学实验教学内容、教学方法、考核方式以及教学反馈进行改革实践,这些改革调动了学生学习的积极性,提高了学生的实践能力与创新思维,教学效果良好.

As for the current teaching mode of medical immunology , it is advisable for educators to explore teaching reform and up-date teaching ideas .In the teaching reform of medical immunology , teachers are expected to find out effective teaching mode through a-nalysis of concrete measures in order to cultivate students ’ scientific thinking and problem-solving ability , and master the basic theoreti-cal knowledge and experiment skills of medical immunology .

日本血吸虫病是由血吸虫引起的一种慢性寄生虫病.本研究着力于探究日本血吸虫(Schistosome japonicum,S.j.)感染的C57BL/6小鼠肠系膜淋巴结不同固有免疫细胞的表型变化.C57BL/6小鼠感染日本血吸虫6周,分离肠系膜淋巴结,制备单细胞悬液,检测固有免疫细胞及其表面相关分子的表达情况.结果显示肠系膜淋巴结中γδT细胞、NK细胞和NKT细胞数量明显增多(P＜0.05),但只有NK细胞的百分比含量明显增加(P＜0.05);γδT细胞、NK细胞和NKT细胞表面CD69表达显著增高(P＜0.01),而CD25的变化均不明显(P＞0.05);γδT细胞和NK细胞表面CD4增高(P＜0.05);NK细胞表面NKG2A/C/E(CD94)、NKG2D (CD314)表达及NKT细胞表面NKG2D (CD314)表达降低(P＜0.05).表明日本血吸虫感染C57BL/6小鼠肠系膜淋巴结不同固有免疫细胞的表型变化存在显著差异.

Liver granulomatous inflammation and fibrosis were the primary pathological changes observed during Schistosoma japonicum (S. japonicum) infection. In the present study, the characteristics of IL-9 were investigated in the liver of S. japonicum infection C57BL/6 mice. Immunofluorescence, qRT-PCR, and ELISA results demonstrated that the expression of IL-9 significantly increased after infection ( P < 0.01). FACS results indicated that the peak of IL-9 + Th9 cells in the liver mononuclear cells appeared at the early phase of infection (week 5), except that Th9 cells, CD8 + Tc cells, NKT and γδT cells could secrete IL-9 in this model. Although IL-9 neutralization has a limited effect on liver granulomatous inflammation, it could decrease the level of fibrosis-associated factor, PC-III, in the serum of infected mice ( P < 0.05). Taken together, our results indicated that IL-9 was an important type of cytokine involved in the progression of S. japonicum infection-induced hepatic damage.

Objective:To study the possible immune escape mechanisms of HCoV-OC43 from human dendritic cells(DC).Methods:HCoV-OC43 was isolated from clinical specimen using BSC-1 cells and identified by Real-time PCR,and the cytopathic effect was observed by phase contrast microscope.DCs were induced in vivo using hu-GM-CSF and IL-4 cytokines,and after 7 days of differentiation,DCs were infected by HCoV-OC43.The morphology of HCoV-OC43 infected DC was observed by transmission electron microscope,and the cytokines related to DC functions were detected by Real-time PCR after infection.DC proportion and function related co-stimulatory molecules were analyzed by flow cytometry.Results:In vitro HCoV-OC43 infected human DC model was successfully built.HCoV-OC43 can infect DC and generate immune response of DC in vitro,but no virus nucleonic acid could be detected in culture supernatant.The DC expression of IFN-α,IFN-β,CCL3 and CCL5 were significant decreased when infected with HCoV-OC43,but the expression of costimulatory molecules including HLA-DR,CD1c and CD86 were not affected by HCoV-OC43 infection.Conclusion:Human DC could be infected by HCoV-OC43 and generate immune response,but could not produce progeny virus.HCoV-OC43 may escape from immune response by suppressing the expression of IFN-α and other inflammatory cytokines and chemokines in DC.

Myeloid-derived suppressor cells (MDSCs), a heterogeneous group of immune cells from the myeloid lineage, play an important part in suppression of host immune responses during many pathologic conditions, including cancer and infectious diseases. Thus, understanding the functional diversity of these cells as well as the underlying mechanisms is crucial for the development of disease control strategies. The role of MDSCs during Schistosoma japonicum infection, however, is not clear, and there is a lack of systematic study so far. In this study, we provide strong evidence that the soluble egg Ag (SEA) and schistosome worm Ag (SWA) of S. japonicum enhance the accumulation of MDSCs. Ag-induced MDSCs have more potent suppressive effects on T cell responses than do control MDSCs in both in vivo S. japonicum infection and in vitro SEA- and SWA-treated mouse bone marrow cells experiments. Interestingly, the enhanced suppressive activity of MDSCs by Ag administration was coupled with a dramatic induction of the NADPH oxidase subunits gp91phox and p47phox and was dependent on the production of reactive oxygen species. Moreover, mechanistic studies revealed that the Ag effects are mediated by JAK/STAT3 signaling. Inhibition of STAT3 phosphorylation by the JAK inhibitor JSI-124 almost completely abolished the Ag effects on the MDSCs. In summary, this study sheds new light on the immune modulatory role of SEA and SWA and demonstrates that the expansion of MDSCs may be an important element of a cellular network regulating immune responses during S. japonicum infection.

Objective:To investigate the influence of Pregnane X Receptor ( PXR) agonist pregnane-16α-carbonitrile ( PCN) on mouse GM-CSF derived dendritic cell ( DC) differentiation.Methods:Mouse DC differentiation was induced by GM-CSF and IL-4 in vitro,and treated with PCN.The proportion of CD11c+DC was measured by flow cytometry;the production of Interferon-γ(IFN-γ), Interleukin-1 (IL-1),Interleukin-2 (IL-2) and Interleukin-12 (IL-12) of DC were analyzed by ELISA and flow cytometry;Real-time RT-RCR was used to detect the expression of genes involved in the signaling pathway which regulated DC differentiation .Results:Mouse DC was successfully induced by GM-CSF and IL-4 in vitro.Treatment of PCN significantly increased the percentage of CD 11c+DC and the protein levels of IFN-γand IL-2 produced by DC were notably enhanced.Real-time RT-RCR results indicated that the expression of Notch and Wnt signaling pathway related genes HES 1,WISP1 and WISP2 were up-regulated in PCN treatment group when compared to control group.Conclusion: The PXR agonist PCN can promote the differentiation of mouse GM-CSF derived DC and increased the production of IFN-γand IL-2 by DC,and Notch and Wnt signaling pathway may play a pivotal role in this process.