【作者】 靳二辉；

【导师】 彭克美； 李奎；

【作者基本信息】 华中农业大学 ， 基础兽医学， 2008， 硕士

【摘要】 五指山猪是我国特有的地方猪种之一,原产于偏僻边远的海南省五指山地区,上世纪80年代引种到北京畜牧兽医研究所,经20多年近交繁育,目前,已培育成近交系数高达0.979的小型猪群体,成年体重仅为30 kg-35kg。近交系五指山小型猪以其体型矮小、遗传稳定、近交程度高等诸多优点,广泛应用于生物医学的各研究领域,成为人类疾病研究理想的动物模型,并列为我国异种器官移植的首选猪种。然而,近些年来,PERV可以在体外感染人细胞的研究发现对猪用于人类异种器官移植的安全性提出了质疑,筛选无PERV的猪用于异种移植变得非常重要。为此,本研究以近交16代的五指山小型猪为研究对象,对其消化系统、泌尿系统和心脏的解剖结构,心脏、肝脏、肾脏、免疫系统和部分生长轴器官的显微和超微结构进行了观察研究,并对其15种组织的PERV-RNA的表达情况进行了检测,主要研究结果如下:1近交系五指山小型猪消化、泌尿器官及心脏的解剖学研究本试验通过大体解剖学方法对近交系五指山小型猪消化、泌尿器官及心脏的解剖特点进行观察,结果显示:近交系五指山小型猪前臼齿发育一般较晚,胃外观呈“V”形,小肠和大肠较其它猪种短,肝脏呈圆角梯形。肾前端较后端钝圆,后端距正中线较远,部分五指山小型猪有两条肾动脉入。肾,膀胱没有明显的膀胱三角。从2-12月龄,心脏横径变化较大,右心耳与腔静脉窦之间界嵴不发达,冠状窦口处没有瓣膜,奇静脉直接开口于此。与人类的相比,舌上叶状乳头发达,轮廓乳头较少,胃贲门和幽门相距较近,空肠和回肠较长,盲肠粗大,结肠形成结肠圆锥;肾脏、膀胱大体形态和位置与人类的相比基本相似,而肝脏和胰腺的形态与人类的相比差异较大。12月龄心脏的重量、体积等解剖常数较成人类的小,左右肺静脉直径较小,前后腔静脉管径一致。这些结构特点表明,近交系五指山小型猪消化系统与人类的相比差异较大,其用于异种器官移植存在解剖学上的困难。而心脏和肾脏与人类的相比较为接近,适用于异种器官移植。2近交系五指山小型猪免疫器官的组织学观察为给近交系五指山小型猪用于建立人类免疫相关疾病模型提供基础形态学方面的资料,本试验采用石蜡切片和HE染色对其免疫器官的组织特点进行观察研究。结果显示:从出生到性成熟,胸腺实质内处于分裂期胸腺细胞和成熟胸腺细胞的数量均增多,胸腺小体的数量也增多。成年时,胸腺细胞数量减少,排列疏松,胸腺小体和胸腺上皮细胞均减少。2月龄时胸腺小体周围可见许多大小不同的空泡状细胞和变形细胞碎片。脾白髓动脉周围淋巴鞘和脾小结在4月龄前也随年龄增长而逐渐增多增大,但12月龄时有所减少并维持在一定水平,其变化与大鼠和鸡的基本一致。2月龄时,淋巴结皮质在内,髓质在外,4月龄时两者分界不明显。与2和4月龄相比,12月龄淋巴小结较大,淋巴细胞排列疏松,髓质内淋巴细胞较少,毛细血管增多。从整体上观察,免疫器官的组织学结构与人和其它哺乳动物之间没有明显的差异。3近交系五指山小型猪心、肝、肾显微和超微结构研究心、肝和肾移植在同种和异种器官移植中都具有重要的地位和作用,研究这3种器官的结构特点对其应用于移植具有重要的意义。本试验通过显微和超微组织学技术对近交系五指山小型猪心、肝和肾的形态特征进行研究,结果表明,2月龄小型猪这些器官结构还没有发育完全,4月龄小型猪各结构非常明显,正处于旺盛的发育期。但从整体角度观察,12月龄时,心室壁、肝脏和肾脏的组织学结构以及心室肌、肝脏和肾皮质的超微结构与人类的最为相近,适合作为异种移植的器官供体。4近交系五指山小型猪垂体、甲状腺和胰腺的形态学研究近交系五指山小型猪脑垂体位于成形的垂体窝内,腺垂体包绕神经垂体,中间部较狭窄,其内滤泡样结构不明显,腺垂体促性腺激素细胞分泌颗粒最大,促肾上促肾上腺皮质激素细胞分泌颗粒最小,神经垂体轴突内分泌颗粒较少,赫令体不明显。甲状腺腺泡随年龄的增长逐渐增大,腺泡上皮细胞微绒毛不发达,腺泡旁细胞有高电子密度和低电子密度两种。胰腺腺泡排列紧密,腺泡间结缔组织不发达,胰岛周围没有结缔组织包绕,相邻腺泡细胞侧面有连接复合体,胞质内充满大而圆的酶原颗粒,泡心细胞较大,部分呈立方形,腺泡间毛细血管可见立方形的胞核。以上结果表明,近交系五指山小型猪脑垂体、甲状腺和胰腺解剖组织学和超微结构与人类和其他哺乳动物的有一定差异。5近交系五指山小型猪PERV的检测采用RT-PCR技术对近交系五指山小型猪15种组织内PERV的表达情况进行了检测,发现PERV在心脏组织中几乎没有表达,在肝脏、肾脏和脑组织中的表达量较低,在免疫、生殖和内分泌组织中普遍存在,尤其在免疫组织中表达量较高,这表明PERv对免疫系统有较强的感染力。更多还原

【Abstract】 Wuzhishan pig is one of the specific local swine species and originally distributed in the remote mountain area of Hainan Province in south China.At present,we had gained minipig group with 0.979 inbreeding coefficient and 30kg-35kg mature body-weight by inbreeding after introduced to the Institute of Animal Sciences in Beijing from 80s,19^th century.Wuzhishan minipig inbred line with much merit such as dwarf body type,stabile heritage and high degree inbreeding is extensively applied to different study domain in biomedical,and becoming ideal animal model about the study of human disease,and included as preferred swine species of organ transplantation in China.However,in recent years,the researches discovered that PERV can infect human cell in vitro,which produced doubt to safe of xenotransplantion from pig to human,so it is becoming important that sieving PERV-free minipig for xenotransplantion.In this study,inbred F16 Wuzhishan minipigs were selected as experimental animal to observe anatomical structure of digestive system,urinary system and heart,microstructure and ultrastructure of the heart,liver,kidney,immune system and partial Growth-Axis organs,and detected expression of PERV-RNA in 15 kinds of tissues.Main results were as follows:1 Anatomical study of heart,digestive and urinary organs in Wuzhishan minipig inbred lineThe anatomic feature of heart,digestive and urinary organs were observed by gross anatomical means in Wuzhishan minipig inbred line,the result showed that development of premolar teeth were later,the stomach was "V"-shape in appearance,the small and large intestine were shorter than other swine species,the liver was ladder-shaped with vaulted angle.Anterior extremity of kidney was blunter than posterior extremity that was distant with median line of body,there are two renal arteries in the partial Wuzhishan minipigs,and the bladder with unsharp vesical triangle.The cardiac broad diameter was conspicuously increased from 2 to 12-months old,the crista terminalis were not conspicuous between right auricle and sinus of venae cavae,the azygos vein was directly opened at the open of coronary sinus.Compared to human,the foliate papillae of tongue were not conspicuous,the calciform papillae were less,the distance between cardia and pylorus was closer in stomach,the jejunum and ileum were longer,the appendix was grosser,colon formed colon-cone.The gross morphous and location of kidney and bladder were similar to the human,but the morphous of liver and pancreas were markedly different with the human.The weight and volume of heart in 12-months old Wuzhishan minipig were less than adult,and left and right pulmonary vein were small,and anterior and posterior vena cava were same.The construction features indicated that the digestive system was markedly different with the human in gross anatomy,which was unsuitable for application of xenotransplantion except the heart and kidney.2 Histological observation of immune organ in Wuzhishan minipig inbred lineThe histological characteristics of immune organs were observed by paraffin section and HE staining,to provide basic morphology data for WZS minipig inbred line,it will be used to established animal model of immunologically disease about human.The results showed that thymus corpuscles,dividing and mature thymocyte were gradually increased in thymus substance from birth to sexual mature.The thymocyte were gradually decreased and loosely disposed,the thymus corpuscles and thymic epithelial cell were also decreased in adult.There were many different-sized bulliform cells and deformed cell debris in periphery of the thymus corpuscle in 2-months-old pigs.Before 4-months-old,the periarterial lymphatic sheath and splenic corpuscle were increased as growth in splenic white pulp,but the gentle reduction and retention on invariably level in 12-months-old pigs,those changes were identical to rat and fowl on the whole.There were indistinct limitation between lymphoid node cortex and medulla in 4-months-old, but in 2-months-old,the cortex was in interior of lymphonode substance,the medulla was in exterior.Compared to 2 and 4-months-old,the lymph nodule was larger,and the lymphocyte were loosely disposed in the cortex,the lymphocyte were less,and blood capillaries were gradually increased in 12-months-old pigs.The histological structure of immune organ were similar to that of human and other mammal on the whole.3 Microstructural and ultrastructural study of heart,liver and kidney in Wuzhishan minipig inbred lineThe transplantatation of heart,liver and kidney is very important role in homotransplantations and xenotransplantation,so it is significant to study construction features of those organs for application of transplantation.The morphological characteristics of heart,liver and kidney were observed by microhistological and ultramicrohistological skills in Wuzhishan minipig inbred line,the results showed that development of many organs were immature in 2-months-old pigs,with clear construction features in 4-months-old pigs.But from general observation,the histological structure of ventricular wall,liver and kidney and the ultrastructure of ventricular muscle, liver and cortex of kidney were very similar to human in 12-months-old pigs,so whom is fit donator for the xenotransplantation.4 Morphological study of hypophysis,thyroid and pancreas in Wuzhishan minipig inbred lineThe hypophysis is located in tectonic hypophyseal fossa,anterior pituitary encase posterior pituitary,pituitary pars intermedia is narrow with unsharp follicle structure, secretary granule is the largest in gonadotroph and the smallest in ACTH cell than other endocrine cell in the anterior pituitary,the secretory granule of neuraxon are less and the Herring body is not obvious in the posterior pituitary.Thyroid follicle is gradually becoming large with growth,microvilli of follicular epithelial cells are undeveloped. There are two kinds of ultimobranchial cells with high or low electron density.Pancreas gland alveolus are tightly disposed with undeveloped connective tissue,there are not connective tissue around pancreatic island,the large and round zymogen granule fill in intracytoplasm of acinous cell with junctional complex on the lateral plane,the cubic centroacinar cell is large,the endothelial cell of blood capillary with cubic nucleus between gland alveolus.Above results indicated that the anatomic,histological and ultrastructure of pituitary,thyroid and pancreas have some discrepancy between human and the Wuzhishan minipig inbred line.5 Detection of PERV in Wuzhishan minipig inbred lineThe expressive status of PERV in Wuzhishan minipig inbred line was detected by RT-PCR,the results showed that the PERV was hardly expressed in heart,and with low expression in liver,kidney and brain.The PERV extensively existed in reproduction and endocrine tissue with high expression in immune tissue,which indicated that there are strong infectivity between the PERV and immune system.更多还原