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Isolation of fish macrophage and its physiology

鱼类巨噬细胞的分离及其生理学

基本信息

批准号：
09660207
负责人：
ENDO Makoto
金额：
$ 2.05万
依托单位：
Miyazaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09660207/
关键词：
Fish Macrophage swim bladder inflammation phagocytosis cytokine ストレス分離

项目摘要

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.

用1 mg/尾的福尔马林灭活大肠杆菌在Hanks‘s平衡液中感染罗非鱼24小时后，从鱼囊中采集中性粒细胞。用超声波处理器对中性粒细胞进行超声处理60秒。为收集炎性巨噬细胞，将中性粒细胞溶血物0.2mL注入鱼囊，注射后4d收集渗入脏器的细胞。涂片后用Giemsa、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，渗出液细胞在L-15培养液25℃中孵育48小时，或用含Ficoll的梯度离心法(梯度为1.065,1500转/分，30分钟)。为了检测巨噬细胞的吞噬功能和各种细胞因子(肿瘤坏死因子-α、白介素1-β、白介素10、白介素12)的水平，用兔抗巨噬细胞免疫球蛋白的方法对巨噬细胞进行了检测。此外，免疫刺激剂如葡聚糖提高了巨噬细胞的生理参数。将罗非鱼饲养1周，使其处于较低的应激状态。对血液皮质醇的分析证实，在自我喂养的情况下，鱼的皮质醇水平低于正常饲养的鱼，这意味着自我喂养为鱼类提供了低压力的条件。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生能力。因此，应激状态似乎会影响巨噬细胞的生理学。