【作者】 沙爱龙；

【导师】 孙虎山； 艾洪滨；

【作者基本信息】 山东师范大学 ， 动物学， 2008， 硕士

【摘要】 头足类软体动物的免疫系统还很不成熟,主要依赖血细胞吞噬、水解酶、氧化酶、活性氧等介导的非特异性免疫抵御病原生物,但头足类体内具有脑啡肽(enkephalin, ENK)、白细胞介素-1(interleukins-1, IL-1)、肿瘤坏死因子(tumor necrosis factor, TNF)等活性肽和细胞因子,对其免疫防御起着重要的调节作用。甲硫氨酸脑啡肽(met-enkephalin, Met-ENK)是一种阿片神经肽。随着阿片肽及其受体功能研究的深入,人们发现阿片肽对心血管系统、消化系统、呼吸系统等都有重要的调节作用。而有关短蛸(Octopus ocellatus)Met-ENK免疫调节机制的研究还未见报道。本文采用免疫组织化学方法对Met-ENK、δ和μ受体在短蛸各组织器官中的分布进行了研究,且采用生化测定的方法研究了不同浓度的Met-ENK对短蛸血淋巴中几种体液免疫因子或自由基的影响,以期为头足类软体动物免疫学研究积累资料。免疫组织化学定位结果表明:(1)在短蛸的脑神经节、足神经节、脏神经节、视神经节、外套神经节、口球、食道、嗉囊、胃、胃盲囊、肠、直肠、前唾液腺、后唾液腺、肝胰脏、鳃、鳃腺、心室、鳃心、肾静脉腺质附属物、卵巢、输卵管腺、外套膜背面、外套膜腹面、腕、腕间膜、漏斗中检测到呈不同阳性反应的Met-ENK分子。(2)在短蛸的脑神经节、足神经节、脏神经节、外套神经节、口球、食道、嗉囊、胃、胃盲囊、肠、后唾液腺、鳃、鳃腺、心室、鳃心、肾静脉腺质附属物、卵巢、输卵管腺、外套膜背面、外套膜腹面、腕、腕间膜、漏斗中检测到呈不同阳性反应的δ受体。(3)在短蛸的食道、胃、外套膜背面、腕间膜中检测到呈不同阳性反应的μ受体。Met-ENK和δ、μ受体在短蛸体内不同部位的分布,表明Met-ENK通过与δ、μ受体结合,可能参与了短蛸神经、消化、呼吸、排泄、感觉、运动和免疫等功能的调节。神经系统分泌的Met-ENK,可能沿神经纤维或血液循环分布到其它组织。而Met-ENK和δ、μ受体在短蛸不同部位分布密度的不同,可能与各器官的功能不同有关。生化测定结果表明:(1)Met-ENK浓度为1.00μg/ml和5.00μg/ml时,对短蛸血清MetAP活力有较弱的抑制作用;Met-ENK浓度为25.00μg/ml时,对短蛸血清MetAP活力有较强的抑制作用。Met-ENK对短蛸血细胞MetAP活力有很强的促进效应,且此效应具浓度依赖性。(2)Met-ENK浓度为1.00μg/ml和25.00μg/ml时,短蛸血细胞CAT活力均高于对照组;3种浓度的Met-ENK,实验组血清CAT活力均低于对照组。Met-ENK浓度为5.00μg/ml和25.00μg/ml时,血细胞H2O2含量均高于对照组,Met-ENK浓度为1.00μg/ml时,实验组血细胞H2O2含量低于对照组;3种浓度的Met-ENK,实验组血清H2O2含量均低于对照组。(3)Met-ENK对不依赖卤素的短蛸血清MPO活力有较弱的抑制效应,且此效应具浓度依赖性。低浓度的Met-ENK对短蛸血细胞MPO活力有很强的促进作用,较高浓度的Met-ENK对短蛸血细胞MPO活力有较弱的促进作用。(4)Met-ENK可使短蛸血清和血细胞中GSH含量减少,可降低短蛸血淋巴的抗氧化能力。(5)Met-ENK对短蛸血清ANAE活力有很强的抑制作用。低浓度的Met-ENK对短蛸血细胞ANAE活力有较强的抑制作用,较高浓度的Met-ENK对短蛸血细胞ANAE活力有很强的抑制作用。总之,Met-ENK对短蛸起着重要的免疫调节作用,其作用机制可能是通过与血细胞表面的受体结合而起作用的。更多还原

【Abstract】 For the cephalopod has no special immunity ability, the cephalopod maily depends on phagocytosis of immunocytes, hydrolytic enzymes, oxidant enzymes and oxygen reactive species to kill phagocyted organisms. Moreover, there are enkephalin, interleukin-1 and tumor necrosis factor participating in the regulation and improvement of the immune processes. Met-enkephalin (Met-ENK) is one of the opioid neuropeptide. By studying the functions of opioid peptide and their receptors, it was found that opioid peptide could regulate cardiovascular system, digestive system, respiratory to some extent. The research on immune regulation of Met-ENK to Octopus ocellatus wasn’t reported before. We studied the distribution of Met-ENK, delta receptor, mu receptor in the organs of Octopus ocellatus with immunohistochemical technique, and studied the impact of Met-ENK to some of the immune factors in the hemolymph with biochemical methods, give help with cephalopod immunology.Using the immunohistochemical technique, nerve cell bodies and fibres immunoreactive to the anti-Met-enkephalin sera were observed in the cerebral ganglia, pedal ganglia, visceral ganglia, inspect ganglia and coat ganglia of Octopus ocellatus. Met-ENK immunoreactivity was also localized in the mouth, esophagus, craw, stomach, stomachic caecus, the epithelium of the intestines, rectum, sialoid gland, liver, branchia, branchia gland, ventricle, branchial heart, kidneys, ovary, fallopian tube, back of mantle, abdomen of mantle, wrist, membrane of wrist, funnel, and immunocytes of Octopus ocellatus. By the same method, delta receptor was detected on the cerebral ganglia, pedal ganglia, visceral ganglia, coat ganglia and in the mouth, esophagus, craw, stomach, stomachic caecus, the epithelium of the intestines, sialoid gland, branchia, branchia gland, ventricle, branchial heart, kidneys, ovary, fallopian tube, back of mantle, abdomen of mantle, wrist, membrane of wrist, funnel and immunocytes of Octopus ocellatus. Mu receptor was localized in the esophagus, stomach, back of mantle, membrane of wrist of Octopus ocellatus. Since Met-ENK and opioid receptors are very abundant in many tissues of Octopus ocellatus, met-Enkephalin was likely to accommodate nerve, assimilation, respiration, exudation, procreate, incretion, sense, movement and immunity of Octopus ocellatus Gray. The met-Enkephalin which was excreted by nervous system maybe transport by nervous fibre, haemal circulation. And the different density of met-Enkephalin, delta receptor, mu receptor may be referable to different function of organ.To study the effects of Met-ENK on immunoregulation of Octopus ocellatus, the content of hydrogen peroxide(H2O2), glutathione (GSH), nitric oxide ( NO) and four kinds of enzymes participating in the immune defence in the haemolymph of Octopus ocellatus were assayed after 1, 5, 25μg/ml Met-ENK were added into the haemolymph. The results were as follows. (1) When the concentration of Met-ENK was between 1.00μg/ml and 5.00μg/ml, the methionine aminopeptidases (MetAP) activities in the serum of Octopus ocellatus were restrained faintishly; when the concentration of Met-ENK was 25.00μg/ml, the methionine aminopeptidases (MetAP) activities in the serum of Octopus ocellatus were restrained strongly. The MetAP activities in the blood cell were enhanced strongly with increasing concentration of the Met-ENK. (2) When the concentration of Met-ENK was between 1.00μg/ml and 25.00μg/ml, the catalase (CAT) activities in the blood cell were enhanced; the catalase (CAT) activities in the serum were restrained. The levels of H2O2 in the serum of Octopus ocellatus were decreased; when the concentration of Met-ENK was between 5.00μg/ml and 25.00μg/ml, the levels of H2O2 in the blood cell of Octopus ocellatus were increased. (3) The myeloperoxidase (MPO) activities which don’t depend on halogen in blood cell of Octopus ocellatus were enhanced greatly at the low concentration of the Met-ENK and enhanced faintishly at the high concentration of the Met-ENK. The MPO activities which don’t depend on halogen in the serum were restrained faintishly after Met-ENK was added. (4) The content of glutathione (GSH) was decreased after Met-ENK was added. (5) Theα-Naphthy Acetate Esterase (ANAE) activities were restrained greatly after Met-ENK was added. Overall, the data strongly suggests an involvement of Met-ENK in the regulation and improvement of the immune processes of Octopus ocellatus.更多还原