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目的:观察表面活性蛋白A(SP-A)对大肠杆菌凝集、生长的作用及单核巨噬细胞对大肠杆菌摄取的影响,探讨SP-A在固有免疫中的作用及机制。方法:①SP-A对大肠杆菌凝集的影响:培养大肠杆菌至稳定期,进行分组:空白对照组、BSA组(BSA 25mg/L)、SP-A组(SP-A 25mg/L)。37℃,200r/min培养,观察大肠杆菌形态。②SP-A对单核巨噬细胞吞噬能力的影响:Ficoll-Hypaque密度梯度离心法分离健康志愿者外周血,分离单核巨噬细胞。实验分为空白对照组、SP-A组(SP-A 25mg/L)。快速瑞氏-姬姆萨复合染液染色,显微镜下观察单核巨噬细胞对大肠杆菌的吞噬并摄像。③SP-A对大肠杆菌生长的影响:培养大肠杆菌至稳定期,实验分为空白对照组、BSA组(BSA 25mg/L)、SP-A组(SP-A 25mg/L)。37℃,200r/min培养6h,每1h末于600nm波长分光光度计进行光密度值(OD)测定,绘制大肠杆菌生长曲线。结果:①空白对照组、BSA组大肠杆菌未发生凝集,SP-A组大肠杆菌发生凝集。②与空白对照组相比,加入SP-A后单核巨噬细胞对大肠杆菌摄取明显增加。③加入BSA后,大肠杆菌生长无明显变化(P>0.05);加入SP-A后,大肠杆菌生长受到明显抑制(P<0.05)。结论:SP-A通过促进大肠杆菌凝集发生,一方面直接抑制大肠杆菌生长;另一方面增加单核巨噬细胞对大肠杆菌的摄取,从而在固有免疫中发挥作用。
OBJECTIVE: To observe the effect of surfactant protein A (SP-A) on the agglutination and growth of Escherichia coli and the effect of monocyte-macrophage on the uptake of Escherichia coli, and to explore the role and mechanism of SP-A in innate immunity. Methods: (1) The effect of SP-A on E.coli agglutination: Escherichia coli was cultured in the stationary phase and divided into blank control group, BSA group (25mg / L BSA) and SP-A group (25mg / L SP-A group). 37 ℃, 200r / min culture, observe the morphology of E. coli. ② The effect of SP-A on monocyte phagocytosis: The peripheral blood of healthy volunteers was separated by Ficoll-Hypaque density gradient centrifugation to separate monocytes and macrophages. The experiment was divided into blank control group, SP-A group (SP-A 25mg / L). Rapid Wright - Giemsa complex dye staining, macrophage monocyte macrophage phagocytosis and imaging. The effect of SP-A on Escherichia coli growth: Escherichia coli was cultured to stable phase. The experiment was divided into blank control group, BSA group (25mg / L BSA) and SP-A group (25mg / L SP-A group). 37 ℃, 200r / min culture 6h, at the end of every 1h 600nm wavelength spectrophotometer OD value determination, draw E. coli growth curve. Results: ①In the blank control group, there was no agglutination of Escherichia coli in the BSA group and the colonization of Escherichia coli in the SP-A group. Compared with the blank control group, monocyte-macrophage uptake of Escherichia coli increased obviously after adding SP-A. (3) After addition of BSA, there was no significant change in the growth of E. coli (P> 0.05). After adding SP-A, the growth of E. coli was significantly inhibited (P <0.05). Conclusion: SP-A can directly inhibit the growth of Escherichia coli by promoting the agglutination of Escherichia coli. On the other hand, SP-A increases the uptake of Escherichia coli by monocyte-macrophage and thus plays an important role in innate immunity.