重组葡萄球菌肠毒素A及其突变体蛋白促淋巴细胞增殖作用的研究

 目的 观察重组葡萄球菌肠毒素A（rSEA）及其突变体蛋白对淋巴细胞增殖、活化作用及对淋巴细胞亚群分群的影响。方法 分离健康人外周血单个核细胞（PBMC）及小鼠脾淋巴细胞悬液，体外经rSEA及其3种突变体（rSEA/H187A、rSEA/H225A和rSEA/D227A）蛋白刺激后，采用噻唑蓝法（MTT）检测人和小鼠脾淋巴细胞增殖水平，采用流式细胞术检测人外周血中T淋巴细胞亚群（CD3⁺、CD4⁺、CD8⁺T细胞）及活化CD4⁺、CD8⁺ T细胞和自然杀伤细胞（NK）的含量。结果 5~20 mg·L^-1的rSEA及其3个突变体蛋白对人PBMC和小鼠脾淋巴细胞均有明显促增殖作用（P<0.05）；经rSEA、rSEA/H225A刺激后的人PBMC中，CD3⁺、CD4⁺、CD8⁺T细胞的含量明显高于对照组，rSEA/H187A能上调CD3⁺和CD8⁺T细胞，而rSEA/D227A只上调CD8⁺T细胞（P<0.05）；rSEA及3种突变体蛋白均能上调活化CD4⁺和CD8⁺T细胞，rSEA、rSEA/H187A、rSEA/H225A能上调NK细胞含量（P<0.05）。结论 rSEA及其3种突变体（rSEA/H187A、rSEA/H225A和rSEA/D227A）蛋白对淋巴细胞均有明显促增殖和活化作用，并可不同程度地上调T淋巴细胞亚群CD3⁺、CD4⁺、CD8⁺ 及NK细胞的含量，为筛选出低毒、高效的升白细胞相关药物候选突变体奠定基础。

Effects of Promoting Proliferation of Recombinant Staphylococcal Enterotoxin A and Mutant Proteins on Lymphocytes

OBJECTIVE To investigate the effects of recombinant staphylococcal enterotoxin A (rSEA) and its mutant proteins on lymphocyte proliferation, activation and lymphocyte subsets. METHODS The human peripheral blood mononuclear cells (PBMC) were isolated from human peripheral blood and splenocytes cells were isolated from mouse. MTT colorimetry was established to determine the lymphocyte proliferation in human PBMC and mouse splenocytes cells stimulated by rSEA and its mutant proteins in vitro. The percents of lymphocyte subsets (CD3+, CD4+, CD8+T cell) and activation CD4+, CD8+T cell and nature killer cells (NK) in PBMC were analyzed by FCM. RESULTS 5-20 mg·L-1 rSEA and its mutant proteins showed the significant effects on promoting proliferation of human PBMC and mouse splenocytes (P<0.05). The percents of CD3+, CD4+ and CD8+ T cells in PBMC treated with rSEA and rSEA/H225A were significantly higher than those of the control group.The rSEA/H187A mutant proteins increased the percents of CD3+ and CD8+ T cells, while the rSEA/D227A mutant proteins only increased the percents of CD8+ T cells（P<0.05）. The percents of activation CD4+ and CD8+ T cells were increased by rSEA and its mutant proteins, as the percents of NK cells were increased by rSEA, rSEA/H187A and rSEA/H225A mutant proteins（P<0.05）. CONCLUSION The significant effects on promoting lymphocyte proliferation and activation of rSEA, rSEA/H187A, rSEA/H225A and rSEA/D227A were confirmed. The percents of lymphocyte subsets and NKC were increased in different degrees by rSEA and its mutant proteins, which establishing the foundation for further screening of low toxicity and more efficient agent mutants.