神经肽Y和β-内啡肽内分泌细胞在鲻鱼肠道中的分布和形态

DISTRIBUTION AND MORPHOLOGY OF NEUROPEPTIDE Y AND β-ENDORPHIN ENDOCRINE CELLS IN THE GUT OF GREY MULLET,MUGIL CEPHALUS L.

  • 摘要: 用链霉亲合素-生物素-过氧化物酶复合物(Strept Avidin-Biotin-peroxidase Complex, SABC)免疫细胞化学方法和兔抗人神经肽Y与兔抗人β-内啡肽抗体对鲻鱼肠道不同部位的内分泌细胞进行鉴别和定位研究.结果显示,这两种神经肽的免疫活性内分泌细胞不同程度地分布在鲻鱼前肠前段和后段、中肠和后肠.神经肽Y免疫活性细胞的形态多样,大多数属开放型细胞,具有胞质突起,少数为封闭型细胞,阳性细胞一般出现在肠褶的中部和近端部.β-内啡肽免疫活性细胞则几乎为封闭型细胞,且定位在肠褶基部.神经肽Y主要分布在前肠前段和后段,分布密度分别为18.7个细胞/mm2和2.3个细胞/mm2,而在中肠和后肠仅少量分布(2).β-内啡肽的分布密度在后肠最高,达31.5个细胞/mm2,其次从前肠前段至中肠顺序递减.还讨论了这两种神经肽在鲻鱼肠道中的生理作用.

     

    Abstract: Neuropeptide Y (NPY), an amidated 36 amino acid peptide and a member of the pancreatic polypeptide family, was first isolated from porcine brain.β-endorphin(β-EP), a member of opioid peptide family, was isolated from the pituitary gland as a fragment of the carboxyl terminal sequence of β-eipotropin consisting of 61 91 amino acids. Biochemical and immunochemical studies have shown that NPY and β-EP are widely distributed in the nervous system (brain and spinal cord),pituitary and retina of vertebrates from fishes to mammals,and involved in a wide array of physiological effects, such as regulation of the secretion of piturtary hormones, control of circadian rhythms, memory processing, food and water intake, regulation of feeding, energy balance and plasma insulin levels. Available information on their distribution in the gastroenteritic tract is much restricted. Chiba (1998) reported the coexistence of sertonin and neuropeptide Y in the gut epithelium of the cloudy dogfish, Scyliorhinus torazame. Zhang et al.(1994) investigated the distribution of β-EP in the pancreas of rabbit. As to the teleost, little information was available. In the present study, the identification and localization of endocrine cells in the various parts of intestinal tract in grey mullet, Mugil cephalus were studied by immunocytochemical method of Strept Avidin Biotin Complex (SABC)and two kinds of rabbit antisera raised against mammalian neuropeptide Y and β-endorphin. The grey mullet Mugil cephalus, used in the present study were collected from Dajing Mariculture Farm in Ronghai, Fujian Province. The fish were 21.5-24.7 cm and weighted 90-225g. They were kept deprived of food in aquarium for 2 days. The fish were vivisected, guts were detached and divided into four blocks induding anterior part of anterior intestine, posterior part of anterior intestine, midgut and posterior intestine. The tissue blocks were fixed in Bouins solution without acetic acid for 8-12h, then dehydrated in a series of alcohol and embeded in paraffin. Sections at a thickness of 6 um in transverse plane were immunocytochemically stained with a commercial kit using the Strept Avidin Biotin Complex (SABC)method. The sections were de waxed, hydrated and incubated in 3% H 2O 2 for 20 min to remove endogenous peroxidase activity. Incubation was performed in the primary antibodies, rabbit anti NPY antibody (1∶1000) and rabbit anti β-EP antibody (1∶400),at 4℃ for 24 h. After three washes in PBS, sections were incubated in the secondary antibody (biotinylated goat anti rabbit immunoglobulin G, 1∶100)for 30 min at room temperature, then rinsed in PBS three times and incubated for 30 min at room temperature with strep avidin biotin peroxidase complex(ABC) The tissue bound peroxidase was visualized for 10-30 min with diaminobenzidine (DAB)method. Immunocytochemistry showed NPY and β-EP immunoreactive cells were localized at varying degrees in the anterior part and posterior part of anterior intestine,midgut and posterior intestine. Immunoreactive cells brown, on a unstained background, were easily identified. NPY immunoreactive cells, majority of which were open type with cytoplasmic protrusion and minority of which were close type, had polymorphi and were mainly localized in the middle and apical part of intestinal fold and minorly in the basic part of intestinal fold. Conversly,β-endorphin immunoreactive cells were almost close type and were mainly localized in the basic part of intestinal fold. NPY were distributed mainly in the anterior part and posterior part of anterior intestine at a density of 18.7 cells/mm2 and 26.3 cells /mm2, respectively. But only a few of NPY cells were found in the midgut and posterior intestine, at a density of 5.1 cells/mm2 and 5.5 cells/mm2, respectively. The highest distribution density of β-endorphin, about 31.5cells/mm, was observed in the posterior intestine, and then, the density decreased sequentially from anterior intestine to midgut, at a density of 26.9, 11.6and 9.1 cells/mm respectively.

     

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