【作者】 王家乡；

【导师】 彭克美；

【作者基本信息】 华中农业大学 ， 基础兽医学， 2010， 博士

【摘要】 鸵鸟是世界上现存体形最大的草食性鸟类,具有很高的饲养价值。雏鸵鸟由于机体的各种器官系统尚未发育完善,抵抗力低,对机体内外环境的变化和病原的侵袭非常敏感,容易发病,从而影响其生长。消化管是营养物质消化吸收的重要场所,它的发育程度决定了动物对营养物质消化吸收的情况,动物对营养物质消化吸收的多少影响动物的生长发育。非洲鸵鸟育雏期较长,在育雏期间,生长发育缓慢,胃肠道疾病严重。为了提高非洲鸵鸟的育雏率,降低胃肠道疾病的发病率,科学家们已经对非洲鸵鸟展开了大量的研究。但是到目前为止,有关非洲雏鸵鸟消化道发育规律的研究,尚未见过报道。Ghrelin是由日本科学家Kojima等人于1999年从小鼠的胃中提纯并鉴定了的一种促生长激素分泌受体的特定的内源性配体的一种脑肠肽,由胃黏膜分泌,能影响动物的摄食、饮水、消化道的活动以及动物体内其它激素的分泌、从而影响动物的生长发育。现已证明哺乳动物Ghrelin为28个氨基酸残基的多肽,禽类的Ghrelin则由26个氨基酸残基组成的多肽。Ghrelin广泛分布于多种动物的胃、回肠、肾上腺、心房、乳房、口腔黏膜、食道、输卵管、脂肪、胆囊、淋巴细胞、肾脏、脾脏、淋巴管、心脏、肺、肌肉、心肌、卵巢、精巢、胎盘、前列腺、胰腺、甲状腺、皮肤、垂体、血管中,并且它的分布随着动物年龄的变化而变化,而到目前为止,未见非洲鸵鸟Ghrelin的研究报道。综合以上两个方面的原因,本课题利用石蜡切片HE染色技术、透射电镜(TEM)技术、组织化学技术、免疫组化技术定位表达(SABC)技术及反转录PCR(RT-PCR)技术等综合手段,从组织、细胞、亚细胞、分子等多层次对雏鸵鸟消化管的形态结构、发育特点,Ghrelin的分布发育规律进行全面、系统的研究,旨在为非洲鸵鸟胃肠道疾病的预防和治疗提供可靠的理论依据。主要研究内容和研究结果包括以下5个方面：1.非洲鸵鸟消化管的形态学特点本试验以90d健康非洲雏鸵鸟为试验动物,通过光镜和电镜样本制作,采用大体解剖、石蜡切片、PAS染色、嗜银染色及HE染色技术,研究雏鸵鸟消化管的形态学结构。结果显示：非洲鸵鸟的消化管由口咽腔、食管、腺胃、肌胃、十二指肠、空肠、回肠、盲肠、结肠和直肠构成。非洲鸵鸟无嗉囊,其口咽腔,腺胃、肌胃、空肠和结肠比其它禽类的发达,特别是结肠,位置占据了整个腹腔后半部的大部分；消化管的组织结构从内到外依次分为黏膜层、黏膜下层、肌层和浆膜层；食管有粗大的皱襞,肌层发达,有发达的食管腺；腺胃分有腺部和无腺部,在有腺部内,腺体由位于固有膜的单管腺和位于黏膜下层发达的复管状腺组成,其中复管状腺由大量的腺细胞、少量的内分泌细胞和黏液细胞构成；肌胃含有肌胃腺,并且黏膜肌层较明显,由内纵肌和外环肌组成；小肠绒毛较长,有分支现象,未见中央乳糜管；十二指肠的固有膜中有发达的腺体和集合淋巴小结,黏膜下层内无十二指肠腺；从十二指肠到回肠,肠绒毛的汇合及分支现象更加明显,固有膜内集合淋巴小结的数量逐渐减少,空肠的绒毛弯曲明显；具有一对发达的盲肠；结肠特别发达,有黏膜皱襞,黏膜上皮为复层柱状上皮,其间夹有杯状细胞,肠绒毛短且发达。非洲鸵鸟消化管的这些形态学特点,决定了非洲鸵鸟对粗纤维具有很强的消化吸收能力。2.雏鸵鸟消化管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,利用大体解剖学、组织化学和形态计量学的方法,研究雏鸵鸟消化管的发育特点。研究结果显示：鸵鸟的体重,消化管的重量和长度随日龄而增加；食管、腺胃和十二指肠的相对重量在90d达到高峰,肌胃、空肠、回肠以及所有大肠的相对重量在45d达到高峰；腺胃单管腺和复管腺的厚度、肌胃腺的高度、肌胃黏膜肌层的厚度、肠绒毛的高度和宽度,肌层的厚度与日龄呈正相关；肠绒毛的高度与隐窝深度的比值在不同肠段,其变化趋势也各有差异：在空肠,从1d-90 d比值逐步减少；而在十二指肠和回肠,从1d-45d比值逐步减少,而从45d-90 d比值逐步增加；小肠绒毛杯状细胞和隐窝杯状细胞的数量从1d-45d随日龄增加而增加,45d-90d随日龄增加而减少。绒毛杯状细胞的数量在1d,空肠最高；45d,回肠最高。隐窝杯状细胞的数量在1d和90d时,回肠最高；45d时,十二指肠最高。这些结果揭示非洲鸵鸟消化道的发育从1d-90d逐步完善；特别是1d-45d,从消化道的重量、长度、肠绒毛的特点以及肠杯状细胞的变化可以看出其发育极不完善。因此,在非洲鸵鸟的饲养管理中,应加强1d-45d的饲养管理,在这阶段应饲喂易消化且营养全面的日粮,这样有助于减少雏鸵鸟消化系统疾病。3.Ghrelin阳性细胞在雏鸵鸟胃肠道的分布及发育规律本研究以1d、45d和90d健康非洲雏鸵鸟为试验动物,利用免疫组织化学SABC技术对非洲鸵鸟消化管内Ghrelin阳性细胞(Ghrelin-ic)的分布、形态学特点以及发育变化进行了研究。结果显示：Ghrelin-ic分布于整个胃肠道；腺胃,大量的Ghrelin-ic位于黏膜下层的复管腺内,少量的阳性细胞位于黏膜层的单管腺内；肌胃,Ghrelin-ic位于黏膜层的黏膜上皮下陷的漏斗状的隐窝上皮细胞内；肠道,Ghrelin-ic数量较胃内少,分布于整个肠道的黏膜层的隐窝和肠绒毛上皮细胞之间,黏膜下层和肌层未见阳性细胞。Ghrelin-ic根据其游离面是否到达腔面可分为两类,即“开放型”细胞和“闭合型”细胞,在腺胃和肌胃,主要是“闭合型”阳性细胞,“开放型”阳性细胞较少,而在小肠和大肠,主要是“开放型”阳性细胞,“闭合型”阳性细胞较少；Ghrelin-ic分布密度总的来说,在腺胃最高,从十二指肠、空肠、回肠、肌胃、盲肠、结肠到直肠分布密度逐步降低；在腺胃,阳性细胞的数量从1-45d逐步增加,并且在45d出现一个峰值；在肌胃和小肠,阳性细胞的数量从1-90d逐步增加,并且在90d出现一个峰值；从90-334d,Ghrelin-ic的数量在胃和小肠都是逐步增加,但差异不显著。以上结果说明：Ghrelin-ic在非洲鸵鸟整个胃肠道内有分布,并且其数量随着机体的发育而变化,总的趋势是：从1-90d,随着日龄的增加而逐步增加。根据这些变化可以推论,Ghrelin对非洲鸵鸟消化管的发育起一定的作用。4.非洲鸵鸟Ghrelin基因的克隆和序列分析根据GenBank中所公布鸡(GeneBank登录号：AB075215)的Ghrelin核苷酸序列,设计合成一对特异性引物,以45d非洲鸵鸟腺胃组织中总RNA为模板,采用RT-PCR法,扩增出Ghrelin成熟蛋白编码cDNA序列,将此扩增产物克隆入pMD18-T载体,进行PCR鉴定及序列测定与分析。结果表明：从非洲鸵鸟腺胃中扩增出了Ghrelin基因,扩增产物为191bp；利用DNAMAN将其转化成氨基酸序列,发现非洲鸵鸟Ghrelin的成熟蛋白,由28个氨基酸残基构成；同一性分析表明,非洲鸵鸟Ghrelin基因与鸡、鸭、鹅、火鸡、鸸鹋、日本鹌鹑Ghrelin基因相应序列的同一性分别为95%、94%、84%、83%、84%、84%和82%；氨基酸同一性与鸸鹋、鸡、鸭、鹅、火鸡和鹌鹑分别为88.9%、73%、69.8%、74.6%、73%和68.3%；基因和蛋白氨基酸同一性分析结果说明Ghrelin是一组在进化上高度保守的蛋白质；利用DNAMAN对Ghrelin进行进化树分析,进一步发现非洲鸵鸟与鸸鹋的亲缘关系最近。非洲鸵鸟Ghrelin成熟蛋白cDNA的成功克隆,为进一步研究非洲鸵鸟Ghrelin全基因结构、基因表达与调控奠定了基础。5.Ghrelin基因在非洲雏鸵鸟胃肠道的表达及发育性变化根据GenBank中所公布鸡(GeneBank登录号：AB075215)的Ghrelin核苷酸序列,设计合成一对特异性引物,以1d、45d和90d健康非洲雏鸵鸟胃肠道组织中的总RNA为模板,采用RT-PCR法,对Ghrelin mRNA在非洲雏鸵鸟胃肠道的表达及发育性变化进行了研究。结果表明：Ghrelin mRNA除在空肠、结肠不表达外,在腺胃、肌胃、十二指肠、回肠、盲肠和直肠均有表达；Ghrelin mRNA在胃和小肠组织中的表达随着日龄的变化而变化：1d时,只在腺胃中表达；45d时,Ghrelin mRNA在腺胃、肌胃及回肠中都有表达,而在十二指肠和空肠中没有表达；90d和334d时,Ghrelin mRNA只在空肠中没有表达,而在腺胃、肌胃、十二指肠和回肠中都有表达。以上结果说明：基因在非洲鸵鸟胃肠道有表达,并且其表达呈发育性变化,这进一步说明了Ghrelin对非洲鸵鸟消化管的发育起一定的作用。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 developmental organ and system, low resistance, the disease inducing by the change of internal and external environment and the invasion of pathogen easily occur in ostrich chicks, thus, their growth were influenced. Nutrient absorption is important at all stages of life.The digestive tract, play significant roles in the final stages of nutrient digestion and assimilation. African ostrich has longer brood time, during the brood time, the growth and development is slow, the gastrointestinal disease is serious. So, in order to improve the brooding rate and decrease gastrointestinal disease rate for African ostrich chicks, some studies have been investigated about the African ostrich, but none have investigated the morphological development of the digestive tract of African ostrich chicks. Ghrelin is a brain-gut peptide that has been isolated as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R) from the rat stomach. Ghrelin is predominantly produced by the X/A-like endocrine cells in the oxyntic mucosa of the stomach. Ghrelin regulates a myriad of physiological functions including appetite regulatory effects, regulation of water intake, gastrointestinal motility and regulation of hormone release and synthesis, thus, it can influence the growth and development in vertebrates. It was proved that ghrelin consists of 28 amino acids in mammals, ghrelin consists of 26 amino acids in the chicken. Ghrelin are found in the stomach, ileum, adrenal gland, heart atrium, breast, mucosa of oral cavity, esophagus, oviduct, fat, gallbladder, lymphocyte, kidney, spleen, lymphatic, heart, lung, muscle, cardiac muscle, ovary, testis, placenta, prostate, pancreas, thyroid, skin, pituitary gland, blood vessel in animals, and the distribution are changed gradually with age. However, there have been no studies on the ghrelin in African ostrich chicks.As mentioned above, the objective of this study was to investigate the morphological structure of the digestive tracts, developmental characteristics, distribution and developmental in ghrelin in the gastrointestinal tract of African ostrich chicks by the methods of paraffin section, HE staining, histochemical, immunohistochemistry(SABC), transmission electron microscopy (TEM) and reverse transcription polymerase chain reaction (RT-PCR) methods. The main contents and the research results are as follows:1. Morphological structure of the digestive tracts of African Ostrich Chicks90 d healthy African ostrich chicks were used in the present study to investigate the morphological structure of the digestive tracts of ostrich chicks by the methods of the gross anatomy, paraffin section, HE staining, PAS staining, AgNOR staining and TEM. The results indicated that the digestive tract consists of pharyngo-oral cavity, esophagus, proventriculus, gizzard, duodenum, jejunum, ileum, cecum, colon, and rectum. African ostrich has not crop. The oral-pharyngo cavity, proventriculus, gizzard, jejunum and colon are more developed than other birds, especially in the colon, the location accounted for most of the latter part of the entire abdominal cavity. The four layers, named in order from the lumen outward, are the mucosa, the submucosa, the muscularis, and the adventitia or serosa. The esophagus has grossus plica, the developed muscularis, developed esophageal glands; there are pars glandularis and pars non-glandularis in the proventriculus and in the pars glandularis, the glandular organ of glandular stomach is made up of simple tubular gland in the lamina propria and compound tubular glands in the submucosa; complex tubular glands composed of a large number of glandular cells, minor endocrine cells and mucous cells; the muscularis mucosa of the gizzard is very obviously, it is composed of internal-longitudinal and external-ring smooth muscles. The villi of small intestine is long and has branches, but it has not central chyle vessel. There are developed intestinal gland and many aggregated lymphoid nodule in the lamina proper of the duodenum. There are not duodenal gland in the submucosa. From duodenum to ileum, it is very obvious that intestinal villi have confluens and branches, the intestinal villi of jejunum is crook and showed a "S" shape, the number of aggregated lymphoid nodule is gradually decreasing; African ostrich has a pair developed cecum and very developed colon, the epithelium mucosae is stratified columnar epithelium, and there are many goblet cells between them, there are mucosa plica, the villi are short and developed. These features of the digestive ducts are possible concerned with food habit, and decide that African ostrich has stronger ability to digest and absorb food.2. Developmental morphology of the digestive tract of African Ostrich Chicks1,45 and 90d healthy African ostrich chicks were selected from the normal group. The development of the digestive tract of ostrich chicks was detailly observed by the gross anatomy and performing histochemistry and morphometry. The results of the present study showed the body weight, length and weight of digestive tract increased with age; the weights of the esophagus, proventriculus and duodenum (relative to the body weight) peaked on day 90, the weights of the gizzard, jejunum, ileum and large intestine (relative to the body weight) peaked on day 45; the thickness of simple tubular gland and compound tubular gland of proventriculus, the gizzard glandular height, the thickness of muscularis mucosa of the gizzard, the villus height and width, and muscle thickness were positively correlated with the age of the birds. The ratio of the villus height to the crypt depth differed among the segments of the small intestine and at the different time points: the ratio of the villus height to the crypt depth in the jejunum decreased from day 1 to day 90, while that in the duodenum and ileum decreased from day 1 to day 45, and increased from day 45 to day 90. The number of goblet cells in the intestinal villi and crypts increased rapidly up to postnatal day 45 and then decreased rapidly between day 45 and day 90. The number of goblet cells in the villi was highest in the jejunum on day 1, and in the ileum on day 45; while that in the crypt was highest in the ileum on days 1 and 90, in the duodenum on day 45. These results suggest that the digestive tract develops gradually from postnatal day 1 to day 90 and that the period up to postnatal day 45 is marked by significant developmental changes in the parameters reflective of the digestive capacity, such as the weight, length, and surface area of the intestine and the number of goblet cells. Therefore, in reared African ostrich chicks, feed management should be enhanced between postnatal day 1 and day 45.3. Distribution and developmental changes in ghrelin-immunopositive cells in the gastrointestinal tract of African ostrich chicks1,45 and 90d healthy African ostrich chicks were selected from the normal group. The distribution, morphological characteristics, and developmental changes of ghrelin-producing cells in the gastrointestinal tract of African ostrich chicks were investigated using immunohistochemistry. The results of the present study showed that Ghrelin-immunopositive (ghrelin-ic) cells existed in all regions of the gastrointestinal tract. In the proventriculus, most of the ghrelin-ic cells were observed at the base of glandular lobuli, and a few ghrelin-ic cells were observed in the basal zone of proventriculus plicae. In the gizzard, ghrelin-ic cells were scattered in the glandular epithelia of crypts. In the duodenum, jejunum, ileum, cecum, colon, and rectum, ghrelin-ic cells were scattered in the epithelia of crypts and villi. Two types of ghrelin cells were found; i.e., closed-type cells and cells with triangular or elongated shapes and with their apical cytoplasmic process in contact with the lumen (open-type cells), indicating that ghrelin cells can be classified into two cell types in the entire gastrointestinal tract. The more numerous ghrelin-ic cells in the proventriculus and gizzard were small and round-shaped, so-called closed-type cells. Few ghrelin-ic cells were elongated-shaped with apical cytoplasmic processes in contact with the lumen, so-called open-type cells. On the other hand, in the duodenum, jejunum, ileum, cecum, colon, and rectum, the more numerous ghrelin-ic cells had triangular or elongated shapes with their apical cytoplasmic processes in contact with the lumen (open-type cells). Few ghrelin-ic cells were round-shaped, so-called closed-type cells. Morphometric analysis revealed that ghrelin-ip cells were localized preferentially in the proventriculus; in the duodenum, jejunum, ileum, gizzard, cecum, colon, and rectum, the cell density gradually decreased. Interestingly, in the proventriculus, the number of ghrelin-ic cells increased gradually after birth until postnatal day 45 and peaked on day 45; in the gizzard and small intestine, the number of ghrelin-ic cells increased gradually after birth until postnatal day 90 and peaked on day 90. There were virtually no differences in the number of ghrelin-ic cells from postnatal day 90 to postnatal day 334 in the stomach and small intestine. By day 45 in the proventriculus and day 90 in the gizzard and small intestine, the number of cells reached a plateau and remained steady. The number of ghrelin-ic cells in the stomach and small intestine was highest in the jejunum and proventriculus on postnatal day 1, but highest in the proventriculus on days 45,90, and 334. These results of the present study clearly demonstrate that ghrelin-ic cells exist and the number of ghrelin-ip cells increases with age in the African ostrich gastrointestinal tract from postnatal day 1 to day 90; ghrelin may be involved in gastrointestinal tract development.4. Cloning and sequence analysis of Ghrelin gene in African ostrich chicksA pair of primers was designed and synthesized according to the gene sequence of chicken Ghrelin(ID:AB075215). RT-PCR method was used to amplify Ghrelin gene from the whole RNA of proventriculus tissue in the 45 d African ostrich chicks. After being cloned in pMD-18T, the PCR segment was determined as Ghrelin cDNA. The results of the present study showed the gene was 191 bp in length and exhibited a high homology with those from chicken (95%), duck(94%), goose (84%), turkey (83%), emu(84%)and Japanese Quail(82%). Mature ghrelins consists of 28 amino acid peptide in African ostrich chicks and exhibited the homology with those from chicken (73%), duck(69.8%), goose (74.6%), turkey (73%), emu(88.9%)and Japanese Quail(68.3%). The result of the present study indicated that Ghrelin gene is highly conservative in evolution according to the homology of the gene and amino acid sequences of bird ghrelin. This study provides a foundation for further analysis of structure, expression and regulation of Ghrelin gene in African ostrich.5. The Distribution and Developmental Changes of ghrelin mRNA Expression in the gastrointestinal tract of African ostrich chicks1,45 and 90d healthy African ostrich chicks were selected from normal group. The distribution, and developmental changes of ghrelin mRNA expressio in the gastrointestinal tract of African ostrich chicks were investigated using RT-PCR. A pair of primers was designed and synthesized according to the gene sequence of chicken Ghrelin(ID:AB075215). And the total RNA of different tissues was extracted to determine the abundance of ghrelin mRNA by RT-PCR. Ninety-day-old female African ostriches were used to investigate the distribution of ghrelin mRNA expressio in various tissues, the results showed ghrelin mRNA existed in the proventriculus, gizzard, duodenum, ileum, cecum, and rectum, there were no expression in the jejunum and colon. Developmental changes in the ghrelin mRNA in the African ostrich stomach and small intestine, the results of the present study showed that ghrelin mRNA existed in the proventriculus, there were no expression in other tissue on day 1; ghrelin mRNA existed in the proventriculus, gizzard, and ileum, there were no expression in the duodenum and jejunum on day 45; ghrelin mRNA existed in the proventriculus, gizzard, duodenum and ileum, there were no expression in the jejunum on day 90 and 334.These results of the present study clearly demonstrate that ghrelin mRNA exist and the distribution of ghrelin mRNA changes with the age in African ostrich gastrointestinal tract from postnatal day 1 to day 90; ghrelin may be involved in gastrointestinal tract development.更多还原