摘要
目的:研制抗结核的新型疫苗(DC疫苗)。方法:无菌分离C57BL/6小鼠骨髓细胞,以rGM-CSF及rIL-4诱导分化获得树突状细胞(DC),以pEGHsp65融合质粒转染DC,共聚焦激光扫描显微镜检测转染率,继以电镜鉴定细胞形态、流式细胞术分析组合性表面分子表达、混合淋巴细胞反应(MLR)检测体外刺激T细胞增殖的功能。将制备的DC疫苗静脉接种正常同系小鼠,分别取各脏器冰冻切片后荧光显微镜观察;取小鼠静脉血用ELISA法检测IL-12及IFN-γ的表达量。结果:24 h DC转染率约20%,电镜可见细胞呈典型毛刺状突起,流式细胞分析表明转染后的DC表面MHCⅡ、33D1的表达量均呈显著增高。MLR显示,pEGHsp65转染DC组与pEG-FP-C1转染DC组及空白DC组比较呈统计学增高。脏器冰冻切片显示,接种pEGHsp65转染DC小鼠组及pEG-FP-C1转染DC小鼠组与未转染DC组比较,在各脏器分布中,以脾脏分布显著增高。其余脏器分布无显著差别。pEGHsp65转染DC小鼠组IL-12及IFN-γ表达量显著高于pEGFP-C1转染DC小鼠组及未转染DC小鼠组,P<0.05,未转染DC组与阴性对照组相比,IL-12及IFN-γ表达量也有统计学增高,P<0.05。结论:pEGHsp65制备的DC疫苗具有成熟表型及功能,能够刺激小鼠IFN-γ及IL-12分泌增高。
Objective： To develop novel vaccine （DC vaccine） against tuberculosis. Methods： Separate marrow of the C57BL/6 mice in asepsis was done, followed by suspend the tissues in medium to induce dendritic cells （DCs） supplemented with rGM-CSF and rIL-4, transfect DCs with constructed pEGHsp65, with rates detected by the laser scanning confocal microscopy （LSCM）. The morphology, phenotype and functional properties were detected by scanning electron microscopy, flow cytometry and mixed lymphocyte reation（MLR）, respectively. The DC vaccines were inoculated into the mice of the same line, afterwards organs were prepared in frozen sections, observing under the fluorescent microscopy; the levels of IL-12 and TNF-7 of the plasm of each group were detected by the ELISAs （Enzyme-linked Immunoassays）. Results： The transfection rate 24h afterwards was around 20%. The electron microscopy showed large veils of the cells. And the FACS showed higher expression of MHC Ⅱ , 33D1 on the surface of the dendritic ceils transfected with pEGHsp65 or pEGFP-C1 than the untransfected dendritic cells did. In the allogeneic mixed lymphocyte reaction, dendritic ceils transfected with pEGHsp65 were more potent to stimulate the proliferation of T cells compared with those transfected with pEGFP-C1 or the untransfected ones. The organ frozen sections manifested that the pEGHsp65-transfected DCs inoculated mice group and the pEGFP-Cl-transfected DCs inoculated group showed broader distribution than the DCs inoculated group, predominantly in the spleen. The pEGHsp65-transfected DCs inoculated mice group showed higher IL-12 and IFN-γ secretion in the plasm than the ones inoculated with pEGFP-Cl-transfected DCs or untransfected DCs, P%0.05. However, the levels of the DCs-inoculated group were higher than that the negative control group, for both IL-12 and IFN-γ P〈 0.05. Conclusion： pEGHsp65-transfected DCs may have mature phenotype and function, which can stimulate mice to produce higher IFN-γand IL-12.
出处
《武汉大学学报（医学版）》
CAS
2005年第6期747-751,i0002,共6页
Medical Journal of Wuhan University
基金
湖北省卫生厅基金资助项目(项目编号:JxlB007)
关键词
树突状细胞
疫苗
结核杆菌
融合质粒
Dendritic Cells
Vaccine
Mycobacterium Tuberculosis
Constructed Vector
