Studies on free radical molecules produced from kuruma shrimp hemocytes

NO在无脊椎动物的软体动物免疫细胞和棘皮动物体腔细胞中均有表达，但在对虾血细胞中未见表达。哺乳动物巨噬细胞经脂多糖刺激产生NO，与干扰素γ(干扰素γ)共刺激可增强其产生NO的能力。NO的产生受到其他炎性细胞因子的影响，这些细胞因子在先天免疫中发挥重要作用，并通过系统发育从无脊椎动物到哺乳动物都是保守的。因此，在对虾血细胞中证明NO的重要性不仅是为了澄清ROI以外的自由基的证据，而且还为探索对虾防御系统中的细胞因子网络提供了工具，在该网络中，细胞防御因子由一种化学介质细胞因子调节(Bogdan等人，2000；Remick和Villarete，1996)。本研究证明了一种潜在的化学发光探针，Cla，专用于超氧和单线态氧，以及在脂多糖和重组人干扰素γ(重组人干扰素γ)共同刺激下，对虾血细胞中NO的产生。对虾血细胞经佛波酯(PMA)刺激后依赖共轭亚油酸的CL被超氧化物歧化酶抑制。用荧光法测定对虾血细胞在脂多糖和重组人干扰素γ共同刺激后产生的一氧化氮(NO)。刺激后的血细胞培养上清液显示荧光强度增加，这种强度随N、G-单甲基-L-精氨酸(L-NMMA)的加入而减弱。这些结果表明，对虾血细胞可以产生O^-2和NO，这些自由基在对虾的防御网络中起着重要作用。

NO has been demonstrated to be in molluscan immunocytes and in echinoderm coelomocytes in invertebrates but not yet demonstrated in shrimp hemocytes. Mammalian macrophage produces NO by LPS stimulation which is enhanced by co-stimulation with interferon γ (IFNγ). NO production is affected by other inflammatory cytokines, which play important roles in innate immunity and are conserved from invertebrate to mammals through phylogeny. Therefore, the importance of demonstrating NO in shrimp hemocytes is not only to clarify the evidence of free-radicals other than ROI, but also to provide the tools for exploring the cytokine network in the shrimp defense system, in which cellular defense factors are regulated by a chemical mediator, the cytokines (Bogdan et al., 2000 ; Remick and Villarete, 1996).The present study demonstrates the potential chemiluminescent probe, CLA, specific for superoxide and singlet oxygen, and the NO production after co-stimulation with LPS and recombinant human interferon γ (rhIFNγ) in shrimp hemocytes.The Cypridina luciferin analogue (CLA), a highly sensitive chemiluminescence (CL) probe, was used to detect superoxide (O_2) in kuruma prawn hemocytes. The CLA-dependent CL of the shrimp hemocyte after phorbol 12-myristate 13-acetate (PMA) stimulation was inhibited by superoxide dismutase. Nitric oxide (NO) production from the shrimp hemocytes was detected by fluorometric measurement after co-stimulation with lipopolysaccharides (LPS) and recombinant human interferon γ in vitro. The supernatant of a hemocyte culture after stimulation showed an increase in the fluorescence intensity, and this intensity decreased with the addition of N^G-monomethyl-L-arginine (L-NMMA). These results show that shrimp hemocytes can produce O^-_2 and NO and these free radicals play an important role in the shrimp defense network.