植物血凝素与金色葡萄球菌肠毒素刺激人外周血单个核细胞的比较研究  

Comparative Study on the Stimulation of Human Peripheral Blood Mononuclear Cells by Phytohemagglutinin and Staphylococcal Enterotoxin A

作　　者：龚超群[1] 郜世隽[2] 蔡继业[1] 黄飞程[1] 

机构地区：[1]暨南大学生命科学技术学院化学系,广东广州510632 [2]暨南大学医学院微生物学免疫学教研室,广东广州510632

出　　处：《分析测试学报》2010年第5期442-448,共7页Journal of Instrumental Analysis

基　　金：国家自然科学基金资助项目(30872404);海外及港澳学者合作研究基金资助项目(30828028)

摘　　要：比较了植物血凝素(PHA)与金色葡萄球菌肠毒素(SEA)对外周血单个核细胞(PBMCs)的刺激效果。取健康人外周静脉血,分离得到PBMCs,分别加入PHA及SEA进行刺激并孵育12 h,利用原子力显微镜和倒置荧光显微镜观察细胞形态变化,利用量子点联合共聚焦显微镜观察活化后细胞膜表面CD3、CD69的分布情况,利用流式细胞术检测淋巴细胞活化早期细胞分化抗原CD69分子表达的变化情况。结果显示,丝裂原PHA刺激的淋巴细胞大多成群聚集,而超抗原SEA刺激的淋巴细胞大多呈分散状,且二者形态有明显差异;两组活化后淋巴细胞的体积均大于静息组,且活化过程中发生极化作用,迁移淋巴细胞,形成膜突起;丝裂原和超抗原刺激淋巴细胞12 h后,使淋巴细胞表达CD69抗原分子,但在量表达上存在差异性(PHA:39.5%±8.7%;SEA:8.3%±1.8%),抗原分子CD3和CD69在膜表面呈不均匀分布,且SEA活化后的T淋巴细胞表面受体CD3和CD69分子在空间上形成了微结构域;外周单个核细胞中其它非T细胞在丝裂原或者超抗原的刺激下也能表达活化抗原分子CD69。The stimulation effects of phytohemagglutinin（ PHA ）and staphylococcal enterotoxin A （SEA） on peripheral blood mononuclear cells（PBMCs） in vitro were compared. PBMCs were isolated from peripheral blood of healthy donors by the Ficoll - Hypaque gradient density method, and were then stimulated and cultured with PHA and SEA for 12 h, respectively. Atomic force microscopy （AFM） and inverted microscope were used to observe the variation of cell morphology ; laser scanning confocal microscopy（LSCM） combined with quantum dots（QDs）was used to observe the distributions of CD3 receptor and activated marker CD69 receptor on the cell membrane surface after activation and flow cytometry （FCM）was used to analyze the expression of the differentiation antigen CD69 on the cell surface in earlier activation period. Results showed that most of the lymphocytes stimulated by mitogen PHA aggregated in groups, whereas those stimulated by the superantigen SEA scattered ; the volume of activated lymphocytes in both groups were greater than that of resting group. During the activation process, polarization and migration of lymphocytes occurred forming protruding membrane. The mean relative amount of CD69 expression after stimulating PHA （ 39.5% ± 8.7% ） were higher than that after stimulating SEA （8.3% ± 1.8% ）. Based on the above results, the activation state and efficiency of lymphocytes, as well as the stimulation features of superantigen SEA were further understood. The results may be helpful for the study of the killing effect of superantigen on tumor cells.

关 键 词：植物血凝素 金色葡萄球菌肠毒素 外周血单个核细胞 激光共聚焦显微镜 原子力显微镜 流式细胞分析仪 量子点 

分 类 号：O766.1[理学—晶体学] Q271[生物学—细胞生物学]