【作者】 岳敏；

【导师】 顾为望；

【作者基本信息】 南方医科大学 ， 人体解剖与组织胚胎学， 2014， 博士

【摘要】 动物的生长是一个复杂的生理过程,受到体内外多种因素的影响。包括动物的品种及性别、营养水平、生活环境、母体效应等。多种因素最终通过神经内分泌的整合、调控而发挥作用,其中调控关键的是由生长激素释放激素-生长激素-胰岛素样生长因子构成的神经内分泌生长轴。神经内分泌生长轴主要包括生长激素释放激素、生长抑素、生长激素、胰岛素样生长因子以及这些激素的受体、结合蛋白。哺乳动物在体内外各种因素的刺激下,下丘脑开始分泌生长激素释放激素,同时也释放生长抑素,生长激素的分泌受到生长激素释放激素和生长抑素的双重控制,即生长激素释放激素刺激生长激素的分泌,而生长抑素抑制生长激素的分泌。生长激素分泌后与生长激素结合蛋白结合经血液循环运输到体内各组织器官,与靶器官上的生长激素受体结合,启动细胞内的信号转导机制,促进胰岛素样生长因子-1的表达。胰岛素样生长因子-1主要由肝脏合成后释放到血液中,再与胰岛素样生长因子结合蛋白结合运输至动物体内的多种组织,促进蛋白质的合成,促进细胞增殖,从而促进哺乳动物以骨骼肌为主的生长。生长激素还作用于肝外组织,使之分泌胰岛素样生长因子,发挥自分泌或旁分泌作用。生长激素在胰岛素样生长因子-1的介导下发挥其促生长作用,一定范围内肝脏中的胰岛素样生长因子-1的分泌随血液中生长激素含量的升高而升高;当血液中的胰岛素样生长因子-1含量超过一定浓度,则会反馈抑制垂体中生长激素的表达,促进下丘脑中生长抑素的分泌,在下丘脑和垂体双重水平上抑制生长激素的合成与分泌。小型猪在分类学上与普通家猪相同,同属于哺乳纲,偶蹄目,不反刍目,野猪科,猪属动物。由于猪和人体在解剖、生理学方面很相似,1700年英国人JohnAuther就曾提出可用猪作为人的循环系统等研究的模型。但家猪体型过大（有些品种成年体重在400kg以上）,使用起来非常不方便。早在二十世纪50年代美国就开始选育适于实验用的小型猪。育成的小型猪在解剖结构、生理学方面和家猪相同,但体型较小,便于科学实验。目前小型猪在生物医学等领域中亦得到了广泛的应用。尤其是在心血管系统疾病、消化系统疾病、内分泌系统疾病、皮肤疾病的研究方面,以及异种器官移植、医疗器械的检测、药物动态代谢的方面,小型猪都是很有利用价值的实验动物。猪齿的牙质和齿像的构造也和人相似,可用于牙科医学实验。我国具有独特而又丰富的小型猪资源,它们在原产地均为长期近亲交配形成的封闭群体,具有体型小、遗传稳定等特点,与国外的多品种杂交小型猪相比,具有明显的优势。以特有小型猪资源为基础,进行封闭繁育,不仅保存了珍贵的品种资源,而且使品种遗传及表型特征更加稳定,更加符合生命科学研究的要求,达到了保种与选育的双重目的。这是我国具有独特的小型猪资源所带来的无可比拟的优势。我国的小型猪品种有:西藏小型猪、五指山小型猪、版纳微型猪、贵州小香猪、广西巴马小型猪和滇南小耳猪等。其中五指山小型猪、版纳小型猪和广西巴马小型猪的生长相关基因已经有人进行了研究,西藏小型猪与生长相关的基因研究较少。西藏小型猪,来源于青藏高原、海拔2500-4300米的农区和半农牧区,是唯一能够适应高海拔气候和以放牧为主的猪种,封闭的地理环境使西藏小型猪保存了非常纯正的品种资源。本课题组于2004年将西藏小型猪从西藏引进至广州,目前已完成风土驯化及实验动物化研究,并开展了相关动物模型、药物实验及转基因克隆等研究。从免疫学、遗传学研究发现,该品系具有独特的免疫相关指标和遗传特征,加上其独特的外形,是一种优良的实验用小型猪品种。西藏小型猪成年体重仅有25-40kg,是正常肉用猪体重的15-20%。本实验就是对西藏小型猪的生长相关性基因GH、GHR、IGF-1基因进行了系统的研究,为西藏小型猪的小体型提供分子理论和实验依据,为进一步“微型化”群体的培育打下基础。本论文研究中:（1）采用了 PCR产物测序的方法进行GH、GHR基因的分型及SNP位点的寻找,并进行不同基因型的部分生长性状比较分析。结果显示:GH基因在检测区域中发现有5个突变位点,分别为A12G、T45C、G84A、G93A、C133T。而GHR基因在检测区域则未发现突变位点。GH基因,T45C突变位点上TC基因型的西藏小型猪的6-8月龄的腹围值较小,G84A突变位点上AA型的西藏小型猪的3-5月龄的体重、体长值较小,G93A突变位点上GG基因型的西藏小型猪的6-8月龄的体长、体高值较小。我们推测在GH基因T45C、G84A、G93A位点突变的TC、AA、GG基因型可能与体型较小有关。（2）克隆得到了西藏小型猪GHR、IGF-1基因的cDNA序列。结果发现:西藏小型猪GHR基因的片段长1912bp,包含编码区1721bp,该编码区编码了572个氨基酸,与版纳微型猪（JF276446.1）、五指山小型猪（DQ422962.1）的GHR基因高度同源达99%。将西藏小型猪GHR基因的编码区与GenBank中搜索到猪的GHR基因（NM₂14254.2）的cDNA序列进行比对分析发现,在1225pb处发生了T→G的突变,该位点的突变引起相应编码氨基酸的变化,由丝氨酸变成了丙氨酸;与五指山猪GHR基因的cDNA序列进行比对分析发现,在1248bp、1287bp处分别发生了G→A的突变,但该位点的突变并未引起相应编码氨基酸的改变;与版纳微型猪相比,在1656bp处发生了T→C的突变,该位点的突变也未引起相应编码氨基酸的变化。所获得的西藏小型猪IGF-1基因的片段包含编码区567bp,该编码区编码了186个氨基酸,与猪（NM₂14256.1）的IGF-1基因高度同源达99%。将西藏小型猪IGF-1基因的编码区与GenBank中搜索到猪的IGF-1基因的cDNA序列进行比对分析发现,在440bp、455pb处发生了G→A、C→T的突变,该位点的突变引起相应编码氨基酸的变化,分别由组氨酸变成了精氨酸、亮氨酸转变成了丝氨酸。具体该位点的突变是否导致蛋白质功能的改变有待于进一步研究。（3）通过实时的荧光定量PCR技术,对西藏小型猪0岁（1日龄）、0.1岁（36日龄）、0.25岁（90日龄）、0.5岁（180日龄）、1岁（360日龄）、2岁（720日龄）、3岁（1080日龄）的心、肝、脾、肺、肾、肌肉、皮肤组织的GH、GHR和IGF-1基因进行相对表达量的测定分析,从而明确GH、GHR和IGF-1基因在西藏小型猪不同年龄阶段和不同脏器中的表达情况。结果显示:在不同脏器的表达中,GH基因在0岁、0.25岁、0.5岁、1岁表达量最低都是肌肉,在0岁、0.5岁时在皮肤中的表达量最高,在0.25岁、1岁时在肺组织中表达量最高,在0.1岁、3岁时表达量最高的是肝脏。GHR基因在0.25、0.5、1、2、3岁的肌肉组织中表达量最低,在0、0.1岁时心脏组织的表达量最低,在1、2岁时表达量最高的是肺,在0、0.25岁的肾脏组织中表达量最高。IGF-1基因在0、0.25、0.5、1、2岁时肌肉组织的表达量最低,在0、0.25、0.5岁时皮肤组织中表达量最高,在0.1、2、3岁时肝脏组织的表达量最高。在不同年龄阶段的表达中,GH和GHR基因在0.1岁时达到峰值,而IGF-1基因在0.25岁时达到峰值。所以西藏小型猪的生长相关基因的表达呈现出明显的时空特异性。（4）构建了西藏小型猪GHR基因的真核表达载体GHR-pIRES2-EGFP转染到西藏小型猪胚胎成纤维细胞中,采用相对定量RT-PCR方法检测IGF-1基因的表达,结果发现:转染了西藏小型猪生长激素受体的西藏小型猪的胚胎成纤维细胞内的胰岛素样生长因子-1出现了表达上调的现象。更多还原

【Abstract】 Animal growth is a complex physiological process,affected by many factors in vivo and in vitro.Including the animal species and gender,nutrition level,living environment,maternal effect etc..Many factors play important role by ultimately integration and regulation of neuroendocrine.And the key of regulation is nerve endocrine growth axis,which is consisted of growth hormone releasing hormone,growth hormone and insulin like growth factor.The neuro endocrine growth axis including growth hormone releasing hormone,somatostatin,growth hormone,insulin like growth factor,hormone receptors and binding proteins.Mammals are stimulated by many factors from internal and external,hypothalamus begin to secrete growth hormone releasing hormone and somatostatin,growth hormone secretion is regulated by both growth hormone releasing hormone and somatostatin.After the combination of growth hormone and growth hormone binding protein,transported to the organs by blood circulation,combined with target organ of the growth hormone receptor.The process initiate the transduction of intracellular signal,promote the expression of insulin like growth factor-1.Insulin like growth factor-1 release into the blood after synthesized by the liver,conbinated with insulin like growth factor binding protein,and then transported to variety of tissues in vivo,promoting protein synthesis,promoting cell proliferation.The combination mainly promoting growth of skeletal muscle of mammals.Growth hormone also play important role in extrahepatic tissues,the expression of insulin-like growth factor by autocrine or paracrine.Growth hormone play growth promoting effect,was mediated by insulin like growth factor-1,The increasing of the liver of insulin like growth factor-1 secretion along with the increases of growth hormone in blood;when the blood insulin like growth factor-1 concentration exceeds a certain concentration,it will inhibit the expression of growth hormone of pituitary,promote the secretion of somatostatin in the hypothalamus.Miniature pig,belong to the class of Mammalia,Artiodactyla,not Pecora,Suidae,pig animal.Because of the swine and human is very similar in anatomy,physiology.In 1700,the British JohnAuther had proposed the swine can be the study model of human circulatory system.But the pig’s oversize（The body weight of some adult swine in 400kg above）,it is very inconvenient.In American,small pig breeding suitable for experimental were bred in early 1950s.The same as the porker in anatomy,physiology,but smaller,more convenient for doing scientific experiment.The widespread application of these miniature pig in the biomedical field,especially in the study of cardiovascular system disease,digestive system disease,endocrine system diseases,skin diseases,as well as xenotransplantation,medical devices for the detection and drug dynamic metabolism research,miniature pig is the value of experimental animal.And it can be used for the experiment of Dental Medicine.The resources of miniature pigs are rich and unique in our country,and are closed colony as long-term inbreeding.They have the characteristics of small size,genetic stability,have obvious advantages compared with foreign varieties of hybrid swine.The resources of The miniature pigs as the base,by blocking breeding,not only saves precious varieties,and the genetic and phenotypic characteristics of varieties is more stable,more in accordance with the requirements of science research,the purposes of conservation and breeding.In China,we have these miniature pig breeds:Tibet min-pig,Wuzhishan miniature pig,Banna miniature pig,Guizhou miniature pig,Guangxi Bama miniature pig and Diannan small ear pig.The study of the growth related genes axis of Wuzhishan miniature pig,Banna miniature pig and Guangxi Bama miniature pig have been done,but few studies on Tibet minipig.Tibet mini-pig,comes from the Qinghai Tibet Plateau.The altitude of 2500-4300 meters farming and farming and pastoral areas,is the only one who can adapt to high altitude climate.The closed geographical environment makes Tibet mini-pig kept very pure breed resources.The research group imported Tibet mini-pig to Guangzhou from Tibet in 2004,the current study on acclimatization and experimental animal has been completed,and carried out the research of related animal model,drug experiment and transgenic cloning.Base on the study of immunology,genetics and its unique shape,Tibet mini-pig breed is excellent experimental animal.The adult weight of Tibet mini-pig is only 25-40kg,15-20%of normal porker body weight.This experiment is the growth related GH,GHR and IGF-1 gene of Tibet miniature pig,it can also provide theoretical basis for molecular breeding of small size Tibet mini-pig.In this studies:（1）We found the SNP sites of GH,GHR gene by the method of PCR products sequencing,and analized the relationship of different genotype and some growth trait.Results:Five single nucleotide polymorphisms（A12G,T45C,G84A,G93A,C133T）were found in exon 2 of GH gene.There was no single nucleotide polymorphisms found in exon 9 of GHR gene.Statistical results show that Tibet-minipig withTC、AA、GG genotype at T45C、G84A、G93A of GH gene maybe related to the smaller body size.（2）The cloned GHR,IGF-1 gene cDNA sequence of Tibet miniature pig.The results showed that:Tibet mini-pig GHR gene fragment was 1912bp,containing the coding region was 1721bp,encoding 572 amino acids,99%homology were compare with Banna miniature pig（JF276446.1）and Wuzhishan miniature pig（DQ422962.1）.Compare with pig（NM₂14254.2）,we found that the mutation of T G at 1225pb,the mutation loci cause changes of amino acids encoded serine→alanine;compare with Wuzhishan mini-pig,the results showed that in 1248bp,1287bp were G→A mutated,but the site did not cause a corresponding change in amino acids;compared with Banna miniature pig,T→C mutation in 1656bp,the site also did not cause the corresponding changes of amino acids.Tibet mini-pig IGF-1 gene fragment containing the coding region of 567bp,the coding region encodes 186 amino acids,99%homology were compare with the pig（NM₂14256.1）.Compare with pig（NM₂14256.1）found that the mutation in the 440bp,455pb,occurred at G→A,C→T mutation,and the corresponding amino acids changed,histidine→arginine,leucine→serine respectively.The mutation of the site changed the protein function or not need to be further studied.（3）Real-time PCR was used to analyze GH,GHR and IGF-1 gene expression of Tibet mini-pig heart,liver,spleen,lung,kidney,muscle,skin tissue in 0 years old（1 days old）,0.1 years（36 days）,0.25 years（90 days）,0.5 years（180 days）,1 years（360 days）,2 years（720 days）,3 years（1080 days old）.The results showed that:three genes expressed in 0,0.1,0.25,0.5,1,2,3,of all ages,and heart,liver,spleen,lung,kidney,muscle,skin of various organs.The GH gene,the highest expression of the skin and the lowest expression of muscle at the age of 0,0.5;the highest expression of lung tissue and the lowest expression of muscle tissue at the age of 0.25,1;the highest expression of liver at the age of 0.1,3.The GHR gene,the highest expression in lung and the lowest in muscle at the age of 1,2;the lowest expression in muscle tissue at the age of 0.25,0.5,3;the lowest expression in the heart at the age of 0,0.1;the highest expression level in the kidney tissue at the age of 0,0.25.The IGF-1 gene,the highest expression in skin and the lowest expression in muscle tissue at the age of 0,0.25,0.5,the highest expression in liver at the age of 0.1,2,3,the lowest expression in muscle tissue at the age of 1,2.In different age period,GH and GHR genes and reached the peak at the age of 0.1,while the IGF-1 gene and reached the peak at the age of 0.25.The expression of Tibet mini-pig growth axis related genes showed a temporal and spatial specificity obviously.（4）The prokaryotic expression vector of Tibet mini-pig’s GHR gene was constructed,named GHR-pIRES2-EGFP,was transfected into Tibet mini-pig’s fetal fibroblasts.The relative quantitative RT-PCR method to detect the expression of the IGF-1 gene.The results showed that:IGF-1 mRNA levels in the cells transfected were upregulated.更多还原