【作者】 王悦；

【导师】 俞诗源；

【作者基本信息】 西北师范大学 ， 动物学， 2010， 硕士

【摘要】 目的观察大鵟( Buteo hemilasius)和长耳鸮(Asio otus)胃肠道、肝的组织结构及胃肠道嗜银细胞的形态分布,检测Bax蛋白(Bcl-2 associated X protein,Bax)、转化生长因子β(β2,β3)(Transforming growth factor-β,TGF-β2 ,TGF-β3)、角质细胞生长因子( keratinocyte growth factor,KGF)和生长素(Ghrelin)在大鵟和长耳鸮胃肠道、肝中的表达,探讨各种蛋白的生物活性作用。方法应用光镜和电镜技术观察大鵟和长耳鸮胃肠道、肝的组织结构,采用整块组织的Grimelius银染法观察大鵟和长耳鸮胃肠道嗜银细胞的分布,应用免疫组织化学方法检测Bax蛋白、TGF-β2、TGF-β3、KGF和Ghrelin在大鵟和长耳鸮胃肠道及肝中的表达,用IPP专业图像分析软件和体视学测量对其表达强度进行定量半定量分析。结果1)大鵟胃肠道的组织结构。大鵟胃可分为腺胃和肌胃两部分,光镜下胃可分为4层,从内向外依次为黏膜层、黏膜下层、肌层和外膜。腺胃腺可分为深、浅两种腺,浅层腺为单管腺,深层腺为复管泡状腺。黏膜肌层较薄,为环行平滑肌。黏膜下层由疏松结缔组织构成,其内分布有大量血管和神经。肌层较薄,由环行平滑肌构成。大肠和小肠无黏膜下层,仅由黏膜层、肌层和外膜构成,黏膜肌层较发达,黏膜肌层除回肠由内环肌和外纵肌2层构成外,肠道其它各段均为纵行肌。肌肉层由内环外纵肌构成。2)长耳鸮胃肠道的组织结构特点。胃肠道分为黏膜层、黏膜下层、肌层和外膜4层。腺胃肌肉层由内纵行和外环行2层平滑肌构成,肌胃肌肉层由内环行、中纵行、外环行3层平滑肌构成。肠绒毛有分支现象。盲肠黏膜上皮有更多的杯状细胞,固有层内淋巴细胞较少。肠段肌肉层无纵形肌,只有环形肌。3)大鵟和长耳鸮胃肠道嗜银细胞的分布。大鵟和长耳鸮腺胃和肠道各段都有嗜银细胞分布,嗜银细胞形态多样,有圆形、椭圆形、锥形、长梭形和不规则形等。腺胃的分布密度远大于肠段,从腺胃到直肠嗜银细胞的分布密度不同。4)大鵟和长耳鸮肝脏的显微结构。大鵟和长耳鸮肝脏外覆以结缔组织被膜,被膜结缔组织在肝门包绕肝动脉、门静脉和肝管,并随肝门管结构由肝门进入肝实质形成小叶间结缔组织,将肝实质分隔成许多肝小叶。小叶中央有一条中央静脉,肝细胞索、肝血窦和狄氏间隙围绕中央静脉呈放射状排列,肝细胞索、肝血窦和狄氏间隙相互交错排列,构成肝小叶的主体。5)大鵟和长耳鸮肝脏的超微结构。大鵟和长耳鸮肝细胞内富含线粒体、粗面内质网、高尔基复合体、糖原和溶酶体。线粒体内嵴结构清楚,呈板状,各嵴平行排列;粗面内质网丰富,多集中在胞核附近,膜上附有大量的核蛋白体颗粒;高尔基复合体由1～2层扁平囊泡和周围的小泡组成,多位于胞核与胆小管之间;胆小管是由相邻肝细胞连接面之间局部胞质及膜凹陷形成的管腔窄小的结构。6)大鵟胃肠道内相关活性物质的表达。大鵟胃肠道呈Bax、KGF、TGF-β2、TGF-β3免疫反应阳性。Bax蛋白主要分布于胃黏膜单层柱状上皮细胞,腺胃深腺单层立方上皮细胞的胞膜、胞质及固有层平滑肌纤维,肠黏膜和肠腺的单层柱状上皮细胞胞膜、胞质内及固有层内。KGF阳性物质分布于腺胃浅层单管腺、深层复管状腺和黏膜的上皮细胞膜,肌胃黏膜上皮和固有层单管腺上皮的细胞膜,肠道固有层的血管、淋巴和平滑肌纤维。TGF-β2和TGF-β3主要集中于浅层单管腺上皮细胞、深层复管状腺上皮细胞和黏膜上皮细胞胞质,肌胃黏膜上皮及固有层单管腺细胞胞质,肠黏膜上皮细胞和肠腺上皮细胞的细胞质。大鵟腺胃和肌胃呈Ghrelin免疫反应阴性,十二指肠、空肠、回肠、盲肠和直肠的黏膜层呈Ghrelin免疫反应阳性,阳性细胞分布于黏膜上皮、肠腺上皮和固有层。7)长耳鸮胃肠道内相关活性物质的表达。长耳鸮胃肠道呈Bax、TGF-β2、TGF-β3免疫反应阳性,Bax蛋白主要分布于胃黏膜单层柱状上皮细胞、腺胃深腺单层立方上皮细胞的胞膜、胞质及固有层平滑肌纤维,肠黏膜和肠腺的单层柱状上皮细胞胞膜及胞质。TGF-β2和TGF-β3主要集中于深腺单层立方上皮细胞及黏膜单层柱状上皮细胞胞质,肌胃黏膜单层柱状上皮细胞及固有层单管腺单层柱状上皮细胞胞质内,肠黏膜、肠腺的单层柱状细胞胞质及黏膜固有层内。Ghrelin主要定位于胃腺单层立方上皮细胞,肠黏膜、肠腺的单层柱状上皮细胞和固有层。8)TGF-β2和Bax在大鵟和长耳鸮肝内的表达。大鵟和长耳鸮肝呈TGF-β2和Bax免疫反应阳性,阳性细胞呈棕色或棕褐色,棕色颗粒分布在细胞质内,肝细胞呈TGF-β2免疫反应阳性,阳性细胞分布在肝细胞胞质和内皮细胞。Bax蛋白阳性细胞分布在肝细胞胞质和门管区中央静脉和动脉,门管区的阳性表达明显强于肝细胞。结论1)大鵟胃肠道的组织结构与其他鸟类基本相似,与长耳鸮相比,大鵟绒毛无分支、无中央乳糜管,二者肌肉层的排列方式也不同。2)大鵟与长耳鸮胃肠道嗜银细胞的分布密度不同,可能与生活环境、食物成分、取食方式、排遗活动以及消化作用等多种因素有关。胃肠道内分泌细胞可能具有内分泌、腔分泌和旁分泌3种分泌方式,且以内分泌和腔分泌为主要方式。3)长耳鴞的肝小叶较大鵟易区分,肝细胞排列比大鵟更规则。大鵟肝细胞内粗面内质网、高尔基复合体、线粒体、溶酶体数目及胆小管面的微绒毛数量均比长耳鴞多,提示大鵟肝细胞比长耳鴞的活跃。4)Bax蛋白在大鵟和长耳鸮胃肠道内均有表达,提示Bax参与胃肠细胞的凋亡,可能是禁食对胃肠黏膜的损伤使得细胞得不到充分的营养供给,发生了凋亡,以此来维持黏膜的正常结构。大鵟和长耳鸮胃部Bax阳性表达的面密度值明显大于小肠,说明禁食对胃的损伤比小肠严重。5)大鵟和长耳鸮胃肠道各段均有TGF-β2和TGF-β3免疫阳性物质分布,且在肠内的表达强弱不完全一致,表明它们在胃肠道不同部位所起的调控作用有差异。推测TGF-β2与TGF-β3参与了黏膜的损伤后修复。6)大鵟和长耳鸮肌胃内未发现Ghrelin免疫阳性细胞分布,可能是禁食使大鵟和长耳鸮肌胃内Ghrelin的分泌量减少的原因。从十二指肠到盲肠Ghrelin阳性表达的面密度值呈递减趋势,提示随着消化能力的减弱,Ghrelin在肠道内的表达减弱,表明Ghrelin可能参与鸟类胃肠功能的调节,其分泌量与消化能力相关。7)TGF-β2在内皮细胞的表达比肝细胞强,原因可能是饥饿使肝细胞受损,导致TGF-β2表达,从而抑制内皮细胞生长。大鵟肝内TGF-β2阳性细胞的面密度值大于长耳鸮,可能是因为禁食对大鵟肝的损伤程度较大,导致TGF-β2的表达较多。Bax蛋白阳性细胞分布在肝细胞和门管区,提示禁食对肝脏的损伤作用导致细胞凋亡。更多还原

【Abstract】 ObjectiveTo explore the histological structure,the distribution characteristic of the argyrophil cells and the expression of Bax,KGF,TGF-β2,TGF-β3 and Ghrelin in gastrointestinal tract of Buteo hemilasius and Asio otus,the histological structure,the distributed density and morphology of the argyrophil cells in the gastrointestinal tract of Buteo hemilasius and Asio otus were observed using microscopy and the silver nitrate method of Gremilus,the expression of Bax,KGF,TGF-β2,TGF-β3 and Ghrelin was investigated by immunohistochemistry combined with stereological method.MethodThe histological structure was observed using microscopy method,the distributed density and morphology of the argyrophil cells were observed using the silver nitrate method of Gremilus,the expression of Bax,KGF,TGF-β2,TGF-β3 and Ghrelin was investigated by immunohistochemistry methods in the gastrointestinal tract of Buteo hemilasius and Asio otus.Their expression intensity was analyzed quantitatively and semiquantitatively by IPP professional image analysis software and stereological method.Results1 ) The histological structure of the gastrointestinal tract of Buteo hemilasius.Stomach is consisted of glandular stomach and muscular stomach,is made up of four concentric layers:which are the mucosa,submucosa,muscle layer and outer membrane.Gastric gland in glandular stomach is consisted of simple tubular glands and compound tubuloacinar glands.The muscularis mucosae and muscle layer of stomach is constructed by circular smooth muscle.Submucosa is consisted of connective tissue,nerve and blood vessel. There is not submucosa in the intestine,it is made of mucosa,muscle layer and outer membrane.Muscularis mucosae is obvious,is composed of longitudinal smooth muscle,but internal-ring and external-longitudinal smooth muscle in ileum;Muscle layer of intestine is made of internal-ring and external-longitudinal smooth muscle. 2)The histological structure of the gastrointestinal tract of Asio otus.It is made up of four concentric layers:which are the mucosa,submucosa,muscle layer and outer membrane.Muscle layer is made of internal-longitudinal and external-ring smooth muscle in glandular stomach,internal-ring intermediate-longitudinal,and external- ring smooth muscle in muscular stomach. Villus of intestine has branch. Mucosa epithelium of caecum is plenty of goblet cell,lymphocytes is poor in the lamina propria. Muscle layer of intestine is made of longitudinal smooth muscle.3) Argyrophil cells in the gastrointestinal tract of Buteo hemilasius and Asio otus.The shape of argyrophil cells is diverse,they are mainly roundness,oval,taper,long shuttle and anomaly.The distributed density of the glandular stomach is more than that of intestine, the distributed density of argyrophil cells is different.4) The microstructure of the liver of Buteo hemilasius and Asio otus. There is a serous membrane covering the liver,connective tissue together with the portal vein,hepatic artery and common hepatic duct stretch deeply into liver parenchyma and divide it into many hepatic lobules surrounding central vein. The irregular hepatic cords or plate and sinusoids line into a radiating shape surrounding the central vein,liver cell cord,liver sinusoid and Disse space staggered each other,constitute the main body of hepatic lobule.5)The ultrastructure of the live of Buteo hemilasius and Asio otus.Mitochondrium,rough endopladmic reticulum,golgi body,glycogen,lysosome are present in abundance in hepatocytes.Mitochondria cristae are parallel arrangement and tabular in shape.The RER are found to be parallel arrays and ribosome are rich in the RER membrane.Golgi’s body located between nuclear and bile canaliculi is composed of one or two layers of squamous capsule and round vesicles.canaliculi are joined by tight junction and desmosomes to form a seal to prevent bile from escaping into the intercellular spaces between hepatocytes.6) The expression of interrelated polypeptides in gastrointestinal tract of Buteo hemilasius.Gastrointestinal tract of Buteo hemilasius appeared Bax,KGF,TGF-β2 and TGF-β3 immunoreactivity.Bax immunopositive cells were mainly distributed in the single-layered columnar epithelial cells of stomach mucos,intestinal mucosa,intestinal glands and the single-layered cuboidal epithelial cells in deep glands of the glandular stomach.KGF immunopositive cells mainly distributed in the epithelial cells of the glandular stomach mucosa,mucosa and simple tubular glands of muscular stomach,the blood vessel,lymph and smooth muscle fiber of the intestine lamina propia.TGF-β2 and TGF-β3 immunopositive cells were found in the cytoplasm of the epithelial cells of stomach mucosa,the glandular stomach deep glands,the muscular stomach simple tubular glands,intestine and intestinal glands.Ghrelin immunopositive cells mainly distributed in the mucosal layer of all intestine segments:duodenum,jejunum,ileum,caecum and rectum.7) The expression of interrelated polypeptides in gastrointestinal tract of Asio otus. Bax immunopositive cells were mainly distributed in the single-layered columnar epithelial cells of stomach mucosa,intestinal mucosa,intestinal glands and the single-layered cuboidal epithelial cells in deep glands of the glandular stomach. TGF-β2,TGF-β3 immunopositive cells were mainly localized at the cytoplasm of the single-layered columnar epithelial cells of the mucosa of the glandular stomach,simple tubular glands of muscular stomach,intestinal mucosa,intestinal glands as well as the single-layered cuboidal epithelial cells in deep glands of the glandular stomach and lamina propria in small intestine.Ghrelin immunopositive cells were mainly distributed in the single-layered columnar epithelial cells of the intestinal mucosa,intestinal glands as well as the single-layered cuboidal epithelial cells in deep glands of the glandular stomach and lamina propia of intestine mucosa.8) The expression of TGF-β2 and Bax in liver of Buteo hemilasius and Asio otus. TGF-β2 and Bax immunopositive cell were found in liver of Buteo hemilasius and Asio otus.The immunoreactive product was shown as brown colored granules in the cytoplasm.Hepatocytes appeared TGF-β2 immunoreactivity,positive cells were detected in the hepatocytes and endothelial cells.Bax-protein-positive cells were detected in the cytoplasm of hepatocytes and portal area in Asio otus liver,Light staining could be detected in the hepatocytes,but the most intense staining was in the central veins and interlobular vein.Conclusion1)Histological structure in the gastrointestinal tract of Buteo hemilasius is similar to other birds, compare with Asio otus, branchy and central chyle vessel were not been found in the intestine villus of Buteo hemilasius, the arrangement of muscular layer between the two is different.2) Diffrence of density of argentaffine cells in the gastrointestinal tract of Buteo hemilasius and Asio otus may be correlated with habitat, food habit, feeding mode, excrement activities and digestion.Argentaffine cells may have endocrine,solinocrine and paracrine functions,and solinocrine and paracrine functions is the main ways.3) Hepatic lobule in Asio otus is easier to distinguish,arrangemengt of hepatocyte is regularer than that of Buteo hemilasius.The number of mitochondrium,rough endopladmic reticulum,golgi body,glycogenlysosome in Buteo hemilasius liver is more than that of Asio otus,we concluded hepatocyte of Buteo hemilasius is activer than that of Asio otus.4) Bax-protein-positive cells were detected in gastrointestinal tract of Buteo hemilasius and Asio otus,It suggests that fasting might be harmful to stomach mucosa and intestinal mucosa. Bax protein participate apoptosis to maintain physiological equilibrium. Surface area density of Bax immunopositive cells in stomach is higher than intestine,It suggests that harm of fasting might be more serious in stomach.5) TGF-β2,TGF-β3 immunopositive cells were found in gastrointestinal tract of Buteo hemilasius and Asio otus,the expression of TGF-β2,TGF-β3 is different,it suggested TGF-β2,TGF-β3 participate restore of mucosa.6) The Ghrelin immunopositive cell was not found in muscular stomach because of fasting.The ghrelin immunopositive cell gradually decreased from the duodenum to the caecum,The expression of ghrelin is concern with the digestion function of gastrointestinal tract. It may control the growth and development in birds gastrointestinal tract .7) Expression of TGF-β2 is highest in endothelial cells.It is suggested injury may lead to expression of TGF-β2,inhibit proliferation of endothelial cells, induce endothelial cells apoptosis and blood vessel neogenesis.Bax-protein-positive cells were detected in the hepatocytes and portal area, fasting might be harmful to liver,injury induce cell apoptosis to maintain the equilibrium of organism.更多还原