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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。
Objective To observe the effects of recombinant staphylococcal enterotoxin A (rSEA) and its mutant protein on lymphocyte proliferation, activation and lymphocyte subsets. Methods The peripheral blood mononuclear cells (PBMCs) and splenic lymphocytes from healthy donors were isolated and stimulated with rSEA and its three mutants (rSEA / H187A, rSEA / H225A and rSEA / D227A) in vitro. (T lymphocyte subsets (CD3 +, CD4 +, CD8 + T cells), and activation of CD4 +, CD8 + T cells in human peripheral blood and flow cytometry Killer cell (NK) content. Results The rSEA and its 3 mutants with 5 ~ 20 mg.L-1 significantly increased the proliferation of both human PBMCs and splenic lymphocytes (P <0.05). After stimulation with rSEA and rSEA / H225A, , RSEA / H187A could upregulate CD3 + and CD8 + T cells, while rSEA / D227A only upregulate CD8 + T cells (P <0.05); rSEA and 3 Mutant protein could upregulate the activation of CD4 + and CD8 + T cells. RSEA, rSEA / H187A and rSEA / H225A could upregulate NK cell content (P <0.05). CONCLUSION: The rSEA and its three mutants (rSEA / H187A, rSEA / H225A and rSEA / D227A) can significantly promote the proliferation and activation of lymphocytes and upregulate the expressions of CD3 +, CD4 +, CD8 + And NK cells, which laid the foundation for the screening of low toxicity and high efficiency leukocyte-associated drug candidate mutants.