Isolation of fish macrophage and its physiology

用1 mg/条福尔马林灭活大肠杆菌汉克斯平衡液感染罗非鱼（Oreochromis niloticus） 24 h后采集鱼鳔中性粒细胞。用超声处理器对中性粒细胞进行60秒的超声处理。为了收集炎性巨噬细胞，将0.2 mL的中性粒细胞细胞溶解物注射到鱼鳔中，注射后4天收集渗出到器官中的细胞。细胞涂片，用吉姆萨、非特异性酯酶和过氧化物酶染色鉴定细胞类型。巨噬细胞占渗出细胞的40%。为了纯化炎性巨噬细胞，将渗出细胞在25℃的L-15培养基中培养48小时或用Ficoll梯度离心（梯度为1.065，转速为1500 rpm，离心30分钟）。两种方法均能使巨噬细胞的纯化率达到90%以上。为了研究巨噬细胞的生理机能，采用ELISA法测定了兔IgG对巨噬细胞的吞噬活性和各种细胞因子（TNF-α、IL-1β、IL-10和IL-12）水平。此外，葡聚糖等免疫刺激物提高了巨噬细胞的生理参数。罗非鱼自喂1周，以降低应激状态。对血液皮质醇的分析证实，自喂鱼的皮质醇水平低于正常喂养的鱼，这意味着自喂给鱼提供了低应激条件。在自食条件下采集的巨噬细胞显示出较高的吞噬活性和细胞因子产量。因此，应激状态影响巨噬细胞的生理机能。

Neutrophils of Tilapia Oreochromis niloticus were collected from the swim bladder 24 hrs after infection with 1 mg/fish of formalin-killed Eschericia coli in Hanks' balanced solution. The neutrophils were sonicated using an ultrasonic processor for 60 sec. To collect inflammatory macrophages, the fish were injected with 0.2 mL of the neutrophil cytolysates into the swim bladders, and cells that exuded into the organ were harvested at 4 days after injection. The cells were smeared and stained with Giemsa, non-specific esterase, and peroxidase stains to identify the cell types. Macrophages occupied 40% of the exudate cells. To purify the inflammatory macrophages, the exudate cells were incubated in L-15 medium 25℃ for 48 hrs or subjected to a gradient-centrifuge procedure with Ficoll (1.065 in gradient, at 1,500 rpm for 30 min). Both the methods enabled to purify the macrophages more than 90%.In order to examine the macrophage physiology, the phagocytic activities and various cytokines levels (TNF-α, IL-1β, IL-10, and IL-12) with a ELISA method using rabbit IgG against them were surveyed on the macrophages. Besides, immunostimulants like glucan raised their physiological parameters of macrophages. Tilapia were kept under self-feeding for 1 week to make lower stressful condition. An analysis on the blood cortisol confirmed the lower cortisol level in the fish kept under the self-feeding than the ones kept under the usual feeding, meaning the self-feeding provides the low stress condition to the fish. The macrophages collected from the fish kept under the self-feeding showed the higher phagocytotic activities and cytokine productivities of macrophages. Thus, it appears that the stressful condition influences the macrophage physiology.