【作者】 刘东武；

【导师】 孙虎山； 艾洪滨；

【作者基本信息】 山东师范大学 ， 动物学， 2006， 硕士

【摘要】 贝类神经系统结构简单,中枢神经也比较分散,缺乏专门的内分泌器官,神经系统也具有内分泌作用;贝类无淋巴细胞而无特异性免疫,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;此外,贝类体内有脑啡肽、IL-1(interleukin-1)、IL-2(interleukin-2)、IL-6(interleukin-6)、TNF(tumor necrosis factor)等活性肽和细胞因子,对其免疫防御起着重要的调节作用。本实验采用免疫组织化学定位的方法在栉孔扇贝(Chlamys farreri)的脑神经节、足神经节、脏神经节、嗅检神经节、肠、直肠、肝脏、口唇、唇瓣、肾脏和血细胞检测到亮氨酸脑啡肽(leucine-enkephalin, L-ENK)分子,在栉孔扇贝的脑神经节、足神经节、脏神经节、嗅检神经节、肠、直肠、胃、肝脏、口唇、唇瓣、肾脏、鳃、外套膜、生殖腺和血细胞检测到δ受体、μ受体和κ受体;L-ENK和δ、μ、κ受体在栉孔扇贝体内的广泛分布,表明L-ENK可能参与了扇贝免疫、消化、呼吸、排泄等功能的调节。另外,采用生化测定的方法研究了不同浓度的L-ENK对栉孔扇贝血淋巴中多种自由基的影响,以及不同浓度的L-ENK对栉孔扇贝血细胞吞噬、包囊、聚集和附贴能力的影响,结果表明:(1)栉孔扇贝血淋巴中过氧化氢酶(catalase,CAT)活性随L-ENK浓度的升高而升高,而血细胞中的过氧化氢(hydrogen peroxide, H2O2)含量随CAT活性的升高而降低。(2)L-ENK对栉孔扇贝血清结构型一氧化氮合酶(constitutive NOS, cNOS)和诱导型一氧化氮合酶(inducible NOS, iNOS)活力起抑制作用,对血细胞cNOS和iNOS活力起诱导作用,不同浓度的L-ENK对血淋巴一氧化氮(nitricoxide, NO)含量、cNOS和iNOS活力的影响不同,L-ENK浓度过高和过低时,均不能最有效地诱导血淋巴中的cNOS和iNOS活力。低浓度和中浓度的L-ENK可使血清中的NO含量增加,而对血细胞NO含量没有影响。(3)L-ENK对不依赖卤素的血清髓过氧化物酶(myeloperoxidase, MPO)活力起促进效应;低浓度的L-ENK对血细胞MPO活力有抑制作用,较高浓度的L-ENK对血细胞MPO活力有促进作用。(4)L-ENK可使扇贝血清和血细胞中谷胱甘肽(glutathione, GSH)的含量显著升高,增强血清和血细胞的抗氧化能力。(5)L-ENK对栉孔扇贝血淋巴酸性α-醋酸萘酯酶(α-Naphthy Acetate Esterase,ANAE)活力有较强的促进作用。(6)血细胞的吞噬能力随L-ENK浓度的升高而升高;更多还原

【Abstract】 The mollusk’s nervous system is simple and sparse. The mollusk hasn’t specialneuroendocrine apparatus and the mollusk’s nervous system has the function of secretion.Being no special immune ability, the mollusk mainly depends on phagocytosis ofimmunocytes, hydrolytic enzymes, oxidant enzymes and oxygen reactive species to killphagocyted organisms. Moreover, there are enkephalin, interleukin-1, interleukin-2,interleukin-6 and tumor necrosis factor participating in the regulation and improvement of theimmune processes.Using immunohistochemical technique, we observed nerve cell bodies and fibres wereimmunoreactive to anti-Leu-enkephalin sera in the cerebral ganglia, pedal ganglia, visceralganglia and osphradium ganglia of C hlamys farreri. Leu-enkephaline(L-ENK)immunoreactivity was also localized in the epithelium of the intestines, rectum, digestivediverticula, labial palps, mouth labia, kidneys and immunocytes ofChlamys farreri. By thesame method, delta-, mu-, and kappa receptors were detected on cerebral ganglia, pedalganglia, visceral ganglia and osphradium ganglia ofChlamys farreri. These receptors werealso localized in the epithelium of the intestines, rectum, stomach, digestive diverticula, labialpalps, mouth labia, kidneys, mantle, gill, gonad and immunocytes ofChlamys farreri, too.Since L-ENK and opioid receptors are very abundant in lots of tissues ofChlamys farreri,Leu-enkephalin may engage in receptor-mediated immunity, digestion, breath and excretionactivities.To study the effects of L-ENK on immunoregulation of Chlamys farreri, the content ofnitric oxide ( NO) , hydrogen peroxide(H2O2), glutathione (GSH) and five kinds of enzymesparticipating in the immune defence system in the haemolymph of Chlamys farreri wereassayed after 1, 5, 50 μg/ml L-ENK were added into the haemolymph. The phagocytosis,encapsulation, conglomeration and adhesive abilities of Chlamys farreri haemocytes wereinvestigated after 1, 5, 50 μg/ml L-ENK were added into the haemolymph, too. The results areas follows. (1) The catalase (CAT) activities in the serum and blood cell were enhanced withincreasing concentration of the L-ENK and the levels of H2O2 in the blood cell of Chlamysfarreri were decreased significantly related in part to increased CAT activities. (2) Theconstitutive NOS (cNOS) and inducible NOS (iNOS) activities changed with increasingconcentrion of the L-ENK. Too high or too low concentration of the L-ENK couldn’t beststimulate the nitric oxide synthase (NOS) activities. Low concentration of the L-ENKincreased plasma levels of NO significantly related in part to increased NOS activities, whileit didn’t affect the levels of NO in the blood cell. (3) The myeloperoxidase (MPO) activities inblood cell of Chlamys farreri were decreased at the low concentration of L-ENK andenhanced at the high concentration of the L-ENK. The MPO activities not depending onhalogen in the serum were enhanced after different concentrations of L-ENK were added. (4)The content of glutathione (GSH) was enhanced after different concentrations of L-ENK wereadded. (5) The α-Naphthy Acetate Esterase (ANAE) activities were enhanced after differentconcentrations of L-ENK were added. (6) The phagocytosis ability of Chlamys farrerihaemocytes were enhanced with increasing concentration of the L-ENK. But theencapsulation activities of Chlamys farreri haemocytes changed with increasing concentrationof the L-ENK. At the concentration of 50 μg/ml, L-ENK stimulated the encapsulationactivities, while at the concentration of 5 μg/ml, L-ENK decreased the encapsulation activities.(7) At the concentration of 1 μg/ml, L-ENK decreased the conglomeration activities ofhaemocytes, but high concentrations of L-ENK have no effect on the conglomerationactivities of haemocytes. At the concentration of 1 μg/ml, L-ENK has no effect on theadhesive activities of haemocytes, and high concentrations of L-ENK decreased the adhesiveactivities of haemocytes.Overall, by binding with opioid receptors, L-ENK can participate in the regulation andimprovement of the immune processes of Chlamys farreri.更多还原