【作者】 王青；

【导师】 曾耀英；

【作者基本信息】 暨南大学 ， 免疫学， 2004， 硕士

【摘要】 目的： 通过观察和研究SARS-CoV X4基因转染T细胞后其活化、增殖、分化、凋亡等生物学行为的改变，探讨SARS-CoV X4蛋白的潜在功能及其作用机制，分析SARS-CoV X4在SARS-CoV致病过程中的作用，为SARS的防治提供理论依据。 方法： 以细胞核转染仪将pEGFP-SARS-CoV X4融合蛋白基因导入T细胞；利用共聚焦显微镜和流式细胞仪观察SARS-CoV X4融合蛋白在T细胞内的表达；经双色免疫荧光染色，检测转染后T细胞CD69表达；~3H-TdR掺入检测T细胞增殖；CBA法检测T细胞细胞因子：AnnexinV及7—AAD双染检测T细胞凋亡率。 结果： 在激光共聚焦显微镜和流式细胞仪下均能通过标识蛋白观察和检测到SARS-CoV X4融合蛋白的表达；SARS-CoV X4转染组的T细胞CD69表达率和经PHA刺激后CD69表达率均高于空载体转染组：经PDB+Ion刺激48h，SARS-CoVX4转染组~3H-TdR掺入量高于空载体转染组；经PDB+Ion刺激6h，SARS-CoV X4转染组IL-2、IL-6、TNF-α和IFN-γ的分泌量高于空载体转染组；SARS-CoV X4转染T细胞后可促进IL-6及TNF-α的分泌；空载体转染组和SARS-CoV X4转染组的T细胞凋亡率没有明显的差别。暨南大学硕士学位论文SARs*COVx4基因转染对外周血T细胞生物学行为的影响结论: PEGFP一SARS一CoV X4能有效地在T细胞中表达，SARS一CoV X4转染后能上调T细胞CD69表达，促进PDB十Ion刺激的T细胞增殖和Thl细胞因子分泌，诱导细胞因子IL一6和TNF一a分泌。上述结果提示SARS一CoV X4蛋白对T细胞的生物学行为有调节作用，推测其机制可能是在转录水平调节免疫相关基因的表达。本研究结果对深入研究SARS一CoV致病机理，为SARS的预防和治疗提供一定的理论依据。更多还原

【Abstract】 Objective:To explore the function of the SARS-CoV X4 protein and the role in SARS pathogenesis, studies were performed on the change of T cells behaviors by transfection of SARS-CoV X4 gene.Methods:T cells were transfected with pEGFP-SARS-CoV X4 expression vector and the green fluorescence of EGFP were detected with flow cytometry and confocal microscopy; The expression level of CD69 on T cells transfected with expression vector with or without PHA stimulation were determinated by flow cytometry; Effects of SARS-CoV X4 transfection on T cell proliferation were evaluated by 3H-TdR incorporation ; The production of cytokines were investigated by Cytometric Bead Assay; Annexin V/7-AAD staining was used to assess percentage of apoptosis of T cells.Results:The expression of SARS-CoV X4-EGFP fusion protein could be detected with flow cytometry and confocal microscopy; The percentage of CD69 expression, the incorporation of 3H-TdR and the production of IL-6 and TNF-a of T cells transfected with SARS-CoV X4 were significantly higher than control group; The percentage of Annexin V single positive cells, which indicate T cells apoptosis, were no difference between two groups.Conclusion:SARS-CoV X4 gene can be expressed efficiently in T cells; The expression percentage of CD69 were higher in SARS-CoV X4 transfection group; The production of IL-6 and TNF-a increased in transfection group; the proliferation were increase in transfection group ; These results suggest that SARS-CoV X4 protein can regulate the behaviors of T cells, we propose that SARS-CoV X4 protein perhaps play an important role in SARS pathogenesis; these data provide useful information about the pathogenesis, prevention and treatment of SARS.更多还原