【作者】 唐丽；

【导师】 彭克美；

【作者基本信息】 华中农业大学 ， 基础兽医学， 2009， 博士

【摘要】 鸵鸟是世界上现存体形最大不能飞行的鸟纲动物,具有很高的饲养价值。雏鸵鸟由于各种生理机能尚未健全,抵抗力低,对机体内外环境的变化和病原的侵袭非常敏感,容易发生应激反应。因此,此阶段下丘脑-垂体-肾上腺轴（HPA轴）功能的完善程度决定着机体的应激反应能力,最终影响雏鸵鸟的生长发育。新城疫（ND）是鸵鸟养殖业中最主要的传染病之一,雏鸟的发病率高达50%。ND作为应激原,进入机体后可引起一系列病理变化,使机体HPA轴处于过度激活或失活状态,进一步加重内环境紊乱,引起病情加重。鉴于此,本研究以雏鸵鸟为试验动物,分正常组（0d、45d和90d）和攻毒组(45d,皮下注射鸡新城疫标准毒株F₄₈E₉),利用HE染色技术、透射电镜（TEM）技术、组织化学技术、放射免疫测定（RIA）技术、免疫组化技术定位表达（SABC）技术及原位缺口末端标记（TUNEL）技术等多种手段,从组织、细胞、亚细胞、细胞因子等方面系统研究雏鸵鸟HPA轴的形态结构、发育特点及ND应激下的调控机理。主要研究内容和研究结果包括以下几个方面:1雏鸵鸟HPA轴的形态结构本试验以1d、45d和90d健康非洲雏鸵鸟为试验动物,通过光镜和电镜样本制作,采用石蜡切片及HE技术,研究雏鸵鸟HPA轴的形态结构。结果显示:雏鸵鸟HPA轴有其自身的特点,其肾上腺结构与其它鸟类的有差异。雏鸵鸟的左右肾上腺形态、大小、位置有一定的差异,左侧肾上腺呈长椭圆形,右侧肾上腺呈三角形或扁椭圆形,较左侧的宽,重。肾上腺实质由肾间组织（皮质）和嗜铬细胞（髓质）交错排列,肾上腺皮质组织分为周围带和中央带,周围带可分为外区（被膜下带）和内区。周围带外区细胞的排列似乎与哺乳动物球状带的相似,内区细胞排列相似于哺乳动物束状带的,中央带细胞排列相似于哺乳动物网状带的,这是鸵鸟肾上腺成带的重要标志。相比于位于肾上腺内层（周围带内区和中央带）的细胞,被膜下带细胞的胞质染色较淡,且含有若干空泡。此外,和中央带细胞不同,被膜下带细胞似乎含有更多的线粒体和较少的脂滴。肾上腺髓质细胞排列成索,含有两种嗜铬细胞,即肾上腺素细胞和去甲肾上腺素细胞。它们均含较丰富的细胞器和大量的分泌颗粒,颗粒表面有膜包裹,内含核芯。其主要区别:肾上腺素细胞的分泌颗粒,其核芯多位于中央,核芯与界膜之间有一狭窄的空晕;去甲肾上腺素细胞的分泌颗粒,其核芯偏于颗粒一侧并贴于界膜上,而使颗粒的另一侧留下一个较大的腔隙。以上结果表明,雏鸵鸟HPA轴的形态结构与其它禽类的有差异,其肾上腺的形态结构与部分哺乳动物的相似。2雏鸵鸟HPA轴的发育特点本研究以1d、45d和90d健康非洲雏鸵鸟为试验动物,利用HE染色技术、TEM技术、RIA技术、免疫组织化学SABC技术及TUNEL技术,研究雏鸵鸟HPA轴的发育特点。研究结果显示:雏鸵鸟HPA轴组织学结构随日龄的增长而不断完善,其在生后早期发育较快,45d时肾上腺皮质分区明显,髓质明显增多,细胞器明显增多。不同日龄雏鸵鸟HPA轴内均存在细胞凋亡现象,但凋亡程度有所不同,1d鸵鸟下丘脑内神经细胞凋亡较多（P＜0.01）,之后凋亡逐渐减少;垂体内细胞凋亡较少,凋亡呈增龄性减少;1d鸵鸟肾上腺内的细胞凋亡较少,45d时凋亡明显增多（P＜0.01）,90d时有所减少。不同日龄雏鸵鸟HPA轴内均可检测到GABA阳性产物,随日龄的增长,其表达量呈增加的趋势,平均积分光密度则在不同器官内呈不同的规律性变化;下丘脑内GABA阳性产物表达量较高,45d时的平均积分光度较高（P＜0.01）;垂体内GABA阳性产物表达量较低,其平均积分光度随日龄逐渐下降;肾上腺内GABA阳性产物表达量较低,阳性信号呈增龄性增强,90d时平均积分光度达到最高（P＜0.01）。雏鸵鸟血清ACTH和Cor水平随年龄增长有明显变化。1d鸵鸟血清ACTH水平在1～45d之间呈上升趋势,之后下降至低于1d的水平（P＜0.01）;雏鸵鸟血清Cor水平在1～45d之间呈下降的趋势,之后渐回升到1d的水平。以上研究结果表明,随着日龄的增长,雏鸵鸟HPA轴的结构和功能不断完善,其在生后早期发育较快。3雏鸵鸟ND应激下的HPA轴调控机理将试验动物随机分为攻毒组和对照组。利用HE染色技术、TEM技术、RIA技术、免疫组织化学SABC技术及TUNEL技术,研究雏鸵鸟ND应激下的HPA轴调控机理。研究结果显示:NDV可引起雏鸵鸟HPA轴发生明显的组织病理学变化,肾上腺病变较严重,下丘脑次之,垂体病变较轻,其病变程度显示了雏鸵鸟HPA轴功能受损程度。雏鸵鸟感染NDV,HPA轴内细胞凋亡数量在病毒感染期间明显高于对照组,提示NDV能诱导雏鸵鸟HPA轴系统发生细胞凋亡;在病毒接种后1d,肾上腺内可检测到大量凋亡细胞,接种5d后凋亡数量显著增加（P＜0.01）,之后凋亡呈下降趋势,肾上腺内细胞凋亡的动态变化反映了雏鸵鸟HPA轴对ND病变的适应和调节。雏鸵鸟感染NDV,HPA轴内GABA表达均下降,肾上腺内GABA阳性产物数量在接种后第1d急剧减少（P＜0.01）,至第3d最低（P＜0.01）,之后随着病毒接种时间的延长,其表达量有所回升,但仍低于与对照组,肾上腺内GABA表达量的消长规律提示GABA可能参与了ND应激下的应激反应过程。雏鸵鸟感染NDV,血清ACTH水平于NDV病毒接种后第1d开始上升,至第5d达峰值（P＜0.01）,之后有所下降,渐趋于正常。血清Cor水平于病毒接种后第1d开始下降,至第7d有所回升,第9d渐趋于正常水平,表明NDV接种后雏鸵鸟血清ACTH和Cor水平的变化与其HPA轴功能的损伤及恢复密切相关。更多还原

【Abstract】 The Ostrich is the biggest, unflyable bird existing in the world, whose significant feeding value has assumed. Because of their incomplete physiological function, low resistance, the stress reaction inducing by the change of internal and external environment and the invasion of pathogen easily occur in ostrich chicks, Thus, the perfect function of hypothalamus-pituitary-adrenal axis (HPAA) can decide the stress ability, growth and development of ostrich chicks. New castle disease (ND) is one of the most serious infection diseases that damage ostrich cultivation. The highest disease incidence of ostrich chicks is 50%. As a stressor, ND could invade the organism and cause a series of pathological changes in body, which induced excessive activation or inactivation of ostrich chicks’ HPA axis, and seriously disturbs the organismic internal environment. In the present study, ostrich chicks were divided into normal group (0d, 45d and 90d) and challenge group (45d) at random. Using the methods of HE staining, transmission electron microscopy (TEM), radioactive immunoassay (RIA), immunohistochemistry (SABC), histochemical method and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), the morphological structure, development characteristics of ostrich chicks’ HPA axis and the regulation mechanism in the stress reaction after artificially challenged with chicken NDV were illustrated. The main contents and research results are as follows:1. Morphological structure of ostrich chicks’ HPA axis1, 45 and 90d healthy ostrich chicks were used in the present study to investigate the morphological structure of ostrich chicks’ HPA axis by the methods of HE staining and TEM. The results indicated that the ostrich chicks’ HPA axis has its own features comparing with other birds. Differences between the left and right adrenal glands were found in shape, size and location. The left adrenal gland is oblong, and the right gland is often triangle exhibiting wider and weighter than the left gland. The main tissue of adrenal glands of ostrich chicks were interdigitation consisted of the interrenal tissue (adrenal cortex) and chromaffin cell (adrenal medulla). The interrenal tissue was divided into a peripheral zone (PZ) and a central inner zone (CZ), and the PZ could be divided into outer area (sub-capsular zone, SCZ) and inner area (IZ). The arrangement of SCZ cells appeared similar to that of zona glomerulosa in mammalian adrenal cortex and also the arrangement of IZ cells resembled that of zona fasciculata, as well as the arrangement of CZ cells appeared like the zona reticularis, suggesting the significant signs of zonation in African ostrich adrenal cortex. The cytoplasm of interrenal cells located beneath the adrenal capsule (SCZ) were stained lighter than that of interrenal cells located inside the adrenal gland (IZ and CZ) and contained several vacuoles. Additionally, unlike CZ cells, SCZ cells contained more mitochondria and lesser lipid droplets. The medulla cells formed cell lines, contained two types of chromaffin cells, i.e. adrenaline cells and norepinephrine cells. Their ultrastructural features appeared characterist of affluent cellular organ and considerable catecholamine-containing secretory granules, both types of granules were membrane-limited. The type I granules possessed an central core, and a confined halo could be found between limiting membrane and core; however, the type II granules had an eccentric core, which polarized one side of secretory granule and sticked limiting membrane, giving a lager lacouna appearance in another side of secretory granule. These results demonstrate there are obvious differences in the HPA axis between ostrich chicks and other poultries and the histological structure of adrenal glands was similar with that of some mammalian.2. Developmental characteristics of ostrich chicks’ HPA axis1, 45 and 90d healthy ostrich chicks were selected from normal group. The development characteristics of ostrich chicks’ HPA axis was detailly observed by HE staining, TEM, RIA, SABC and TUNEL. The results showed the HPA axis’ histological structure acquired development unceasingly with the ages of ostrich chicks, especially in early postnatal development. The significant signs of zonation, an evident increase of adrenal medullary cells and cell organs were observed in 45d African ostrich adrenal glands. Apoptosis cells in HPA axis could be detected in any developmental period from 1d to 90d ostrich, but the apoptosis degree varying in different organs. In hypothalamus, A large number of apoptosis cells could be observed at 1d (P<0.01), then gradually decreased. A less number and an age-related decline of apoptosis in pituitary could be found. In adrenal gland, a significant apoptosis peak appeared at 45d (P<0.01), then reduced at 90d. The positive signals of GABA could be detected in HPA axis, with the expression increased with age growing of ostrich chicks. However, the average optical density of positive signals presented various regular change in different organs. The expression of GABA positive products in hypothalamus was stronger, and higher average optical density (P<0.01) could be found at 45d. In pituitary, the expression of GABA positive products was relatively weaker, and an age-related drop of average optical density could be detected. The positive signals in adrenal gland gradually increased along with the increasing of age, and the average optical density reached a peak at 90d (P<0.01). The levels of ACTH and Cor in serum changed with ages of ostrich chicks. 1d ostrich showed higher serum ACTH level, gradually increased with age growing which reached maximum levels at 45d (P<0.01), then dropped to lower than that of 1d (P<0.01). While, the levels of Cor in serum ostrich chicks appeared a decreasing trend from 1 d to 45d, come to the lowest point at 45d (P<0.01), then gradually recovered to the levels at 1d. These results demonstrate that the structure and function of HPA axis in ostrich chicks is under continuous perfection and development, especially in early postnatal period.3. The stress-mediated mechanism of ostrich chicks’ HPA axis with challenged with NDVThe experimental animals were divided into challenge and control groups at random. The methods of HE staining, TEM, RIA, SABC and TUNEL were used to study the stress-mediated mechanism of ostrich chicks’ HPA axis with challenged with NDV. The results are as follows: NDV could cause obvious pathological change of ostrich chicks’ HPA axis in which the adrenal gland was relatively serious than that of the hypothalamus and pituitary, and the pathological degree can demonstrate the damaged degree of HPA axis’ function. After artificially infected with NDV, the apoptosis in HPA axis of challenge group obviously increased, which indicated that NDV can induce the apoptosis of HPA axis of ostrich chicks. In adrenal gland the number of apoptosis increased significantly at 1 day after challenged, and reached maximum at 5 days (P<0.01), then delined. The dynamic changes of apoptosis in adrenal gland indicate that the adapting and regulating of HPA axis in the pathological stress reaction. The expression of GABA positive cells in HPA axis of ostrich chicks in challenge group reduced significantly compared with control group. The amount of GABA positive cells in adrenal gland decreased sharply at 1 day after challenged (P<0.01), reached minimum at 3 days (P<0.01), then increased again. The changes of GABA positive cells in adrenal gland indicate that GABA participate HPA axis’ stress reaction in NDV pathological conditions. The level of ACTH in serum slightly increased at 1 day after challenged, reached its maximum at 5 days (P<0.01), then came back. Whenas, the level of serum Cor always increased until 7 days after challenged, then gradually became normal at 9 days. These results demonstrate that the variation of ACTH and Cor in serum of challenge group correlates closely with the damage and restoration process of HPA axis.更多还原