【作者】 陈敏；

【导师】 彭克美；

【作者基本信息】 华中农业大学 ， 基础兽医学， 2014， 博士

【摘要】 鸵鸟从非洲引进到我国饲养,其生存的气候和环境等均发生了较大的变化,导致出现诸如免疫力下降、生产性能降低等现象。此外,鸵鸟育雏期长达3个月,在育雏期内,雏鸵鸟的生长发育较为缓慢,而且易发生胃肠道疾病。为了提高鸵鸟育雏期的免疫力,降低胃肠道疾病的发生,需要对鸵鸟胃肠道和免疫器官的形态发育与功能调控开展详细的基础研究。Somatostatin (SS)中文译名生长抑素,是Brazeau等人1973年从绵羊下丘脑提取物鉴定的一种神经肽。SS广泛产生于中枢神经和许多外周器官包括胃肠、胰腺、胸腺等器官。SS的功能复杂多样,参与神经功能、激素分泌、免疫反应、胸腺发育、胃肠分泌和运动的调节。目前,对SS在鸵鸟胃肠道和胸腺内的分布和增龄性变化未见研究报道。本研究以此为切入点,采用石蜡切片、免疫组织化学方法和分子生物学技术等综合技术手段,对SS在非洲雏鸵鸟胃肠道和胸腺内的分布定位和增龄性变化规律进行系统的研究,旨在为阐明SS对雏鸵鸟胃肠道和胸腺的功能调控,提供形态学依据,从而为鸵鸟的饲养管理和疾病防治提供理论指导,并为丰富SS在不同种类动物的分布资料、深刻理解SS神经肽功能的多样性和生物进化性提供科学依据。主要工作与研究结果如下：1非洲雏鸵鸟胃肠道内SS的分布表达采用免疫组化SABC法和RT-PCR法分别对90d非洲雏鸵鸟胃肠内SS免疫阳性细胞的分布、形态学特点和SS mRNA的表达进行观察和检测。免疫组化研究结果显示：雏鸵鸟腺胃、小肠内均见有SS阳性细胞的分布,而在肌胃、大肠内未观察到有SS阳性细胞的分布。在腺胃黏膜下层内的腺小叶内,可见有大量的SS阳性细胞分布。在小肠各段的黏膜层内可见有SS阳性细胞分布。在十二指肠内可观察到大量的阳性细胞分布,而在空肠和回肠内,仅有少量的阳性细胞稀疏分布。RT-PCR法研究结果显示：雏鸵鸟的腺胃、小肠和结肠内有SS mRNA表达,而肌胃、盲肠和直肠内未见SS mRNA表达。鸵鸟胃肠道内SS的分布与对其它动物的报道相似,但也存在差异,反映了SS在不同动物胃肠道的分布存在保守性和多样性。2SS免疫阳性细胞在非洲雏鸵鸟腺胃和小肠内发育性变化采用免疫组化SABC法和图像分析技术对1d、45d、90d非洲雏鸵鸟腺胃和小肠内SS免疫阳性细胞的分布进行半定量分析,并与1y鸵鸟比较。结果显示：与1d相比,雏鸵鸟腺胃内SS阳性细胞分布密度值45d时显著升高(P<0.05),于45d达到峰值,90d和1y时又开始逐渐降低；雏鸵鸟十二指肠内阳性SS细胞分布密度值1d时最低,45d和90d时显著升高(P<0.05),于90d达到峰值,1y时又开始降低；而雏鸵鸟空肠和回肠内SS细胞分布密度1d时最高,45d和90d时显著降低(P<0.05)。SS阳性细胞分布密度在育雏期阶段鸵鸟腺胃和小肠内呈现规律性变化,提示SS可能在育雏期鸵鸟胃肠道发育和功能方面,起到重要的调控作用。3非洲雏鸵鸟胸腺内SS的分布表达采用免疫组织化学SABC和RT-PCR法分别对90d的雏鸵鸟胸腺内SS免疫阳性细胞的分布、形态学特点和SSmRNA的表达进行观察和检测。免疫组化试验结果显示：在鸵鸟的胸腺内有少量的SS免疫阳性细胞存在。阳性细胞稀疏地分布在胸腺皮质和髓质区,大多分布在皮质髓质交界处,细胞大多呈圆形或椭圆形,有些细胞呈梭形或梨形细胞,并且伸出细长的突起。RT-PCR检测结果显示：雏鸵鸟的胸腺内有SS mRNA表达。这些结果表明SS可能通过自分泌和旁分泌的方式,参与调节鸵鸟胸腺细胞的发育。4SS在非洲雏鸵鸟胸腺内的发育性变化采用免疫组织化学SABC法和图像分析技术对1d、45d、90d的非洲雏鸵鸟胸腺内SS免疫阳性细胞的分布进行观察分析,并与1y鸵鸟比较。结果显示：雏鸵鸟胸腺内SS阳性细胞分布密度值1d时较低,45d时显著增加(P<0.05)。45d之后,鸵鸟胸腺内SS阳性细胞分布密度值虽有增加,但无显著性差异。这变化表明,SS可能在育雏期前期对鸵鸟胸腺细胞的发育起到重要的调控功能。5非洲鸵鸟SS cDNA克隆和分析根据GenBank中报道的鸡SS mRNA序列设计引物,以非洲鸵鸟腺胃总RNA为材料,采用RT-PCR法,扩增出非洲鸵鸟SS cDNA产物,将其克隆、鉴定及序列测定与分析。结果显示：从非洲鸵鸟腺胃中扩增出了SS基因,序列大小为348bp；用DNAMAN软件将非洲鸵鸟SS基因序列与从GeneBank上查到的其它动物的基因序列进行同源性分析,同源性在95.1%-72.2%之间。使用DNASTAR软件对非洲鸵鸟SS cDNA序列分析显示该核苷酸序列编码116个氨基酸残基的前体肽,将SS前体肽的氨基酸序列与小鼠、大鼠、鸡、斑胸草雀、猪、人、猕猴的相比,不同动物之间的SS前体肽序列主要在信号肽段存在差别。非洲鸵鸟成熟SS-14肽序列与其它动物完全相同,SS-28肽序列与鸡的完全相同,而与啮齿类和哺乳类相比,仅在N-端第9个位点存在一个氨基酸的差异。用DNASTAR软件构建SS分子进化树分析显示：在已经报道物种的SS基因序列中,非洲鸵鸟与鸡之间的亲缘关系是最密切。这些研究结果显示在生物进化上SS基因和蛋白质序列高度保守。更多还原

【Abstract】 The climatic environment and food source hase changed after the ostrich has introduced in our country from abroad, which lead to declined immunity and decreased porfermance to the ostrich. Furthermore, the ostrich, the biggest and unflyable bird living in the world, has longer brood time. During the brood time, the growth anddevelopment of the ostrich is slow, whereas its gastrointestinal disease is serious. So, in order to improve the immunity and decrease gastrointestinal disease rate for ostrich, basic research on morphological development and functional regulation should be investigated in indetail for the ostrich. Somatostatin (SS), a neuropeptide with an inhibitory role on the release of growth hormone, was first isolated in ovine hypothalamic extracts by Brazeau ea al in1973. Studies have demonstrated that in addition to the hypothalamus, SS-immunoreactive (SS-IR) cells also are distributed across other brain regions and in many peripheral organs such as the gastrointestinal tract (GIT), the pancreas and the thymus. SS is involved in a variety of activities including the regulation of neuronal function, hormonal secretion, cardiovascular function, as well as the gastrointestinal function and immune response. The anatomical and histological structure of the GIT and the thymus of the ostrich chicks has been studied and described. However, there are no studies so far on distriubtion of SS in the GIT and the thymus of this bird. The aim of the present study was to investigate the distribution, expression and age-related chanes of SS in the GIT and the thymus of the African ostrich chicks, by immunohistochemistry and RT-PCR, for a better understanding of the physiological role of SS in the GIT tract and the thymus of the ostrich. Moreover, comparative study of distribution of SS in the different animals would help clarify its evolutionary complexity. The works and the results of this study are as follows:1The distribution and expression of SS in the GIT of the African ostrich chicksThe distribution and morphological features of SS-IR cells and expression of SS mRNA were investigated in the GIT of the African ostrich chicks on day90by using immunohistochemical techniques and RT-PCR. Immunohistochemical results show that SST-IR cells were distributed in the proventriculus, duodenum, jejunum and ileum. However, no immunoreactivity was observed in the gizzard, cecum, colon and rectum. In the proventriculus, great numbers of the SS-IR cells were clustered in the base of glandular lobule. These cells had a small round or oval shape. In the small intestinal, SST-IR cells were present in the mucosal layer of whole small intestine of the ostrichs.The number of SST-IR cells were moderate in the duodenal portion but then became rare in the jejunal and ileal portions.SST-IR cells were present in the crypts and villi, which often had round and spherical shapes(closed-type cells). Spindle and pyriform shaped cells, with an apical cytoplasmic process, in contact with the lumen, were also found. The RT-PCR results confirmed the expression of SS mRNA in the proventriculus, duodenum, jejunum, ileum and colon, but no expression of that in the gizzard, cecum and rectum. There are both similarity and difference between our results on the distribution of SS-IR cells in the GIT of the ostrich and study on other animal, suggesting^the distribution of SS-IR cells in the GIT are both conservative and diverse.2The age-related changes of SS-IR cells in the proventriculur and small intestinal of the African ostrich chicksThe development changes of the SS-IR cells in the proventriculur and the small intestinal were detected by immunohistochemical SABC and Image analysis in the African ostrich chicks aged on1day,45days, and90days, and compared with that in African ostrich chicks aged on1year. The results show:The distribution density of SS-IR cells increased significantly on day1, compared with that on day45(P<0.05) in the proventriculus, peaked on day45. However, it decreased significantly on day90, which show no significant difference between these birds aged day90and year1.The distribution density of SS-IR cells increased significantly on day1to day90(P<0.05) in the duodenum, and peaked on day90, which show no significant difference between these birds aged day90and year1. In contrast, it decreased gradually from day1to day90(P <0.05), which show no significant difference between this birds aged day90and year1. There are the regular changes of the SS-IR cells dencity in the proventriculus and small intestine of the ostrich during brood time. SS may be involved in the development and function of the GIT of the ostrich chicks. 3The distribution and expression of SS in the thymus of the African ostrich chicksThe distribution and morphological features of SS-IR cells and expression of SS mRNA were investigated in the GIT of the African ostrich chicks on day90by using immunohistochemical techniques and RT-PCR. Immunohistochemical results show that immunoreative cells were sparely distributed in both the cortical and medullary regions of the thymus, but mainly in the corticomedullary junction. The SS of these cells had a small round or oval shape Spindle and pyriform shaped cells with multicellular processes were also found. Furthermore, The RT-PCR results confirmed the expression of SS mRNA in the ostrich thymus. These findings suggest that SS may play a role in T-cell development via autocrine and paracrine pathways4The age-related changes of SS-IR cells in the thymus of the African ostrich chicksThe development changes of the SS-IR cells in the thymus intestinal were detected by immunohistochemical SABC and Image analysis in the African ostrich chicks aged on1day,45days, and90days, and compared with that in African ostrich chicks aged on1year. The results show. The distribution density of SS-IR cells increased significantly on day1to day45(P<0.05); There was no significant difference after day45. These results suggest SS may play a role in T-cell development during brood time.5Molecular cloning and sequence analysis of cDNA encoding SS from African ostrichThe cDNA encoding SS from African ostrich were firstly amplified from the total RNA of the proventriculur by RT-PCR. The cDNA were cloned, Identified, sequenced and analyzed. The results show as fellow:the nucleotide sequence was348bases. The identities between the sequence of ostrich with that of chicks, zebra finch, pig, dog, cattle, goat, human, Rhesus monkey, crab-eating macaque, mouse, rat, western clawed frog and Chinese sturgeon were among95.1%-72.2%. The translation of the nucleotide sequences into the amino acids was performed using the DNASTAR software. The deduced encoded protein was116amino acids residue protein precursors in length. Compared with the amino acid sequence of other species, the mature SS-14peptide sequence of the ostrich was idenctical with the homologue of other species. SS-28peptide sequence of the ostrich was identical with the homologue of the chicks and had only a residue discrepancy at the9th site of the N-terminal with the homologue of the rodents and the Mammali. The relationship between the African ostrich SS and chicks SS gene was close in the phylogenetic tree analysis by DNASTAR software. These fingings suggest the sequence of aminao acid and nucleotide of SS is highly-conserved in the evolution of life.更多还原