树鼩IgG纯化鉴定及其多克隆抗体制备和检测

目的:比较两种抗体纯化方法在分离纯化树鼩IgG抗体的应用,制备抗IgG的多克隆抗体及检测。方法:采用两种商品化IgG抗体纯化试剂盒分离树鼩血清IgG抗体,采用SDS-PAGE和蛋白定量测定提纯IgG。以树鼩IgG作为抗原,与等量弗氏完全佐剂(第一次)、弗氏不完全佐剂(第二次)混合皮下注射免疫兔,对分离血清进行多克隆抗体纯化及Western Blot检测及定量分析。结果:两种方法均能有效分离纯化树鼩IgG,在经过Montage PROSEP-A试剂纯化后的IgG在纯度和含量方面均优于Protein A/G Matrix试剂。通过纯化后的树鼩IgG免疫兔制备的抗IgG抗体能有效识别树鼩IgG。结论:纯化的树鼩IgG具有良好免疫原性,由此制备的抗体具有高度特异性。研究结果为利用树鼩作为实验动物提供了必要的实验基础。     

Cloning of full-length coding sequence of tree shrew CD3E and prediction of its molecular characteristics

The use of tree shrews (Tupaia belangeri) in human disease studies demands essential research tools, in particular cellular markers and their monoclonal antibodies for immunological studies. Here we cloned the full-length cDNAs encoding CD3E from total RNA of the spleen, liver and peripheral blood of tree shrews and analyzed their structural characteristics in comparison with other mammals by Discovery Studio software. The results showed that the open reading frame sequence of tree shrew CD3E was 582 bp, encoding 194 amino acids. The overall structure of tree shrew CD3E protein was similar to its counterparts of other mammals, intracellular and transmembrane domain highly conserved. However, detailed analysis revealed two potential glycosylation sites and different surface charges in the extracellular domain. Availability of the entire open-reading-frame and related sequence information would therefore facilitate the preparation of monoclonal antibodies against tree shrew CD3 and further studies for its function.     

自发性树鼩乳腺肿瘤的特性(英文)

乳腺癌是严重危害女性健康的常见恶性肿瘤,建立合适的乳腺癌动物模型对于研究人类乳腺癌的生物学机制及发展新的防治方法至关重要。相对于常用的啮齿类动物,树鼩(Tupaia belangeri chinensis,tree shrew)因在进化层次上更接近于人类而可用于建立更适合的乳腺癌模型。该文详细了介绍一例树鼩自发性乳头状良性乳腺肿瘤。免疫组化结果显示该例肿瘤孕激素受体阳性且Ki-67阳性细胞比例显著增加;而活化的Caspase3阳性细胞比例较低;且肿瘤的形态和病理与人导管内乳头状肿瘤非常接近。提示利用树鼩建立乳腺肿瘤模型的可行性。     

北京地区实验树晌的饲养管理方法

树鼩作为低等灵长类动物,在生物学研究中的价值日益引起研究者重视,将其作为实验动物的实验需求越来越多。作为南方亚热带动物,树嗣如何适应北方地区的生活是北方地区将其用作实验动物的前提。本实验室探索出适宜树购居住的饲养笼具,确定了饲养环境温度、湿度、光照时间、噪声及通风等条件,制定了规范化的卫生管理标准,建立了北京地区长期人工培育树晌的饲养管理方法,为今后利用树嗣进行中医药病证结合动物模型的研究奠定了基础。     

Creating animal models,why not use the Chinese tree shrew (Tupaia belangeri chinensis)?

The Chinese tree shrew (Tupaia belangeri chinensis),a squirrel-like and rat-sized mammal,has a wide distribution in Southeast Asia,South and Southwest China and has many unique characteristics that make it suitable for use as an experimental animal.There have been many studies using the tree shrew (Tupaia belangen) aimed at increasing our understanding of fundamental biological mechanisms and for the modeling of human diseases and therapeutic responses.The recent release of a publicly available annotated genome sequence of the Chinese tree shrew and its genome database (www.treeshrewdb.org) has offered a solid base from which it is possible to elucidate the basic biological properties and create animal models using this species.The extensive characterization of key factors and signaling pathways in the immune and nervous systems has shown that tree shrews possess both conserved and unique features relative to primates.Hitherto,the tree shrew has been successfully used to create animal models for myopia,depression,breast cancer,alcohol-induced or non-alcoholic fatty liver diseases,herpes simplex virus type 1 (HSV-1) and hepatitis C virus (HCV) infections,to name a few.The recent successful genetic manipulation of the tree shrew has opened a new avenue for the wider usage of this animal in biomedical research.In this opinion paper,I attempt to summarize the recent research advances that have used the Chinese tree shrew,with a focus on the new knowledge obtained by using the biological properties identified using the tree shrew genome,a proposal for the genome-based approach for creating animal models,and the genetic manipulation of the tree shrew.With more studies using this species and the application of cutting-edge gene editing techniques,the tree shrew will continue to be under the spot light as a viable animal model for investigating the basis of many different human diseases.     

Tree shrew (Tupaia belangeri) as a novel laboratory disease animal model

The tree shrew (Tupaia belangen) is a promising laboratory animal that possesses a closer genetic relationship to primates than to rodents.In addition,advantages such as small size,easy breeding,and rapid reproduction make the tree shrew an ideal subject for the study of human disease.Numerous tree shrew disease models have been generated in biological and medical studies in recent years.Here we summarize current tree shrew disease models,including models of infectious diseases,cancers,depressive disorders,drug addiction,myopia,metabolic diseases,and immune-related diseases.With the success of tree shrew transgenic technology,this species will be increasingly used in biological and medical studies in the future.     

树鼩(Tupaia belangeri)淋巴细胞的自体花结

参与人自体花结(A花结)形成的分子(如CD2/LFA-3),与免疫细胞的粘附和激活有关。我们曾发现,人和猴淋巴细胞表面的树鼩红细胞(TRBC)受体不同于绵羊红细胞(SRBC)受体(CD2),可能是一种新的白细胞分化抗原。花结试验表明,树鼩的外周血淋巴细胞(TPBL)和胸腺细胞都能形成A花结,结花率分别为20.9％和11.1％;而绵羊红细胞花结(E花结)形成率分别是20.9％和1.1％。以四种单克隆抗体(McAb)(Leu 5,0-275,AICD2.1和E2 McAb)进行树鼩A花结和E花结的抑制与抗原调变试验,结果表明,这些抗体对树鼩的A花结都没有明显的抑制或调变作用,但对E花结的抑制及调变作用明显。说明TPBL表面的TRBC受体不同于SRBC受体,与CD2/LFA-3及E2分子无关。因此,TPBL的A花结与E花结形成机制不同。     

树鼩载脂蛋白E的cDNA和蛋白质结构分析

以从树肝脏mRNA逆转录得到的Ⅰ链cDNA为模板 ,运用SMARTRACEPCR技术 ,扩增得到树载脂蛋白E(apoE)cDNA序列 ,并推导出apoE蛋白质的氨基酸序列 .利用分子生物学软件包PCGENE对氨基酸序列和二级结构进行分析和比较 .结果表明 ,树apoEcDNA序列 (作为新基因已被GenBank接收 ,登录号为AF 30 3830 )由 1138bp构成 ,其中 5′非翻译区 6 4bp ,3′非翻译区 135bp ,939bp组成一个完整开放阅读框架 ,与人apoEcDNA的同源性为 86 % .编码 313个氨基酸组成的apoE前体 ,包含 18个氨基酸构成的信号肽和 2 95个氨基酸组成的成熟蛋白 .与人apoE氨基酸序列的同源性为 78% .树apoE与人及其它种属动物apoE在氨基酸组成上相近 ,但比人apoE少4个氨基酸 ,比动脉粥样硬化易感动物家兔apoE多 2个氨基酸 .经Garnier法预测 ,树apoE蛋白二级结构与人apoE相似 ,螺旋构象 (helical) 6 9 9% ,伸展构象 (extended) 16 6 % ,转角构象 (turn)6 0 % ,无规则卷曲 (coil) 7 6 % .     

人工饲养树鼩血液学及生化指标正常值测定及分析

目的建立人工饲养健康树鼩血液学和血液生化指标的正常值参考范围,并探讨与不同物种的血液学和血液生化值的差异及相互关系。方法应用全自动血细胞仪和生化分析仪测定140只健康成年树鼩的血液学及血液生化指标。结果雌性与雄性比较,LYM％、PLT、RDW％、GLU、CREA的差异具有显著性（JP〈0．05）,11P、CHOL、TG的差异具有极显著性（P〈0．01）,其他指标的差异不具显著性,部分指标与恒河猴、大鼠及人的相应指标存在一定的差异。结论本文建立了健康树鼩的血液学及生化指标的正常值参考范围,可为应用该动物进行科学研究时提供参考。     

Repetitive sequences of the tree shrew genome (Mammalia, Scandentia)

Copies of two repetitive elements of the genome of common tree shrew (Tupaia glis) were cloned and sequenced. The first element, Tu III, is a approximately 260 bp long short interspersed element (SINE) with the 5'-end derived from glycine RNA. Tu III carries long polypurine- and polypyrimidine-rich tracts, which may contribute to the specific secondary structure of Tu III RNA. This SINE was also found in the genome of smooth-tailed tree shrew of another genus (Dendrogale). Tu III seems to be confined to the order Scandentia (tree shrews) since it was not found in DNA of other tested mammals. The second element Tu-SAT1 is a tandem repeat with a monomer length of 365 bp. Some properties of its nucleotide sequence suggest that Tu-SAT1 is a centromeric satellite.     

树鼩应激镇痛的研究

用辐射热和电刺激两种方法测定了应激状态下树鼩(Tupaia belangeri chinensis)的痛反应值。结果表明,在应激状态下,树鼩的痛反应值在1—7分钟内下降了100—200％。上述结果提示,树鼩的痛反应降低与应激镇痛有关。     

Proteomic characteristics of the liver and skeletal muscle in the Chinese tree shrew (Tupaia belangeri chinensis)

Valid animal models are useful for studying the pathophysiology of specific disorders, such as neural disease, diabetes and cancer. Previous molecular phylogeny studies indicate that the tree shrew is in the same order as (or a close sister to) primates, and thus may be an ideal model in which to study human disease. In this study, the proteome of liver and muscle tissue in tree the shrew was identified by combining peptide fractionation and LC-MS/MS identification. In total, 2146 proteins were detected, including 1759 proteins in liver samples and 885 proteins in skeletal muscle samples from the tree shrew. Further sub-source analysis revealed that nearly half of the identified proteins (846 proteins and 418 proteins) were derived from human database. In this study, we are the first to describe the characteristics of the proteome from the liver and skeletal muscle of the tree shrew. Phylogenetic tree analysis based on these proteomic data showed that the tree shrew is closer to primates (human) than to glires (the mouse and rat).     

树鼩乳汁的主要成分检测及其意义

目的：探讨树鼩乳汁的基本成份,并和其它乳制品成份进行了比较分析。方法选取10只分娩后（1-21）d期间的哺乳期母树鼩,用人工被动母乳喂养方法让仔树鼩自由吮吸母乳,立即采用无菌操作的方法用注射器直接采取仔树鼩胃内的乳汁,1次／2 d,连续3-5次,每只母树鼩采取18 mL乳汁,按照国家标准的方法进行成分检测。结果树鼩乳汁总固形物为43.63％、脂肪为26.01％、蛋白质为10.41％、乳糖为0.45％、灰分为0.99％。树鼩乳的总固形物、灰分、蛋白质、脂肪、乳糖含量分别为牛乳的3.36、1.24、2.74、6.67、0.09倍;树鼩乳的总固形物、灰分、蛋白质、脂肪、乳糖含量分别为婴幼儿配方乳的1.44、0.20、0.58、1.53、0.06倍;与牛乳及婴幼儿配方乳矿物质成分含量相比,树鼩乳的矿物质成分钙、磷、钾、钠、镁、铁、锌含量均大于牛乳,分别为1.83、2.73、1.25、1.93、1.28、1.48倍,而均小于婴幼儿配方乳,分别为0.66、0.85、0.34、0.26、0.85、0.24、0.49倍。结论树鼩乳的主要营养成分呈现高脂高蛋白低糖的类型,可为树鼩人工育幼和饲养工作提供依据。     

树鼩资源的开发利用与标准化研究——我国实验动物资源建设发展战略探讨

树鼩（Tupaia belangeri）作为人类疾病动物模型,被应用于人类医学实验研究的多个领域,如各种类型的病毒性肝炎模型,流感病毒、登革热病毒、轮状病毒、疱疹病毒模型,糖尿病、胆结石、癌症病模型。但是,目前我国用于实验研究的树鼩几乎全都来自野外,对它们的微生物学、寄生虫学、遗传学、环境学等质量标准没有进行控制,它们本身携带的许多病原体会对实验结果的准确性造成影响,这既不符合国家对实验动物许可证管理的要求,也不利于我国实验动物科技工作与国际接轨的发展要求。因此,应该同步开展树鼩资源的开发利用和标准研究,使之纳入实验动物许可证的有效监控和管理,改变目前使用在前、质量控制标准研究在后的被动局面。     

Defining the ancestral karyotype of all primates by multidirectional chromosome painting between tree shrews, lemurs and humans

We used multidirectional chromosome painting with probes derived by bivariate fluorescence-activated flow sorting of chromosomes from human, black lemur (Eulemur macaco macaco) and tree shrew (Tupaia belangeri, order Scandentia) to better define the karyological relationship of tree shrews and primates. An assumed close relationship between tree shrews and primates also assists in the reconstruction of the ancestral primate karyotype taking the tree shrew as an ”outgroup” species. The results indicate that T. belangeri has a highly derived karyotype. Tandem fusions or fissions of chromosomal segments seem to be the predominant mechanism in the evolution of this tree shrew karyotype. The 22 human autosomal painting probes delineated 40 different segments, which is in the range found in most mammals analyzed by chromosome painting up to now. There were no reciprocal translocations that would distinguish the karyotype of the tree shrew from an assumed primitive primate karyotype. This karyotype would have included the chromosomal forms 1a, 1b, 2a, 2b, 3/21, 4–11, 12a/22a, 12b/22b, 13, 14/15, 16a, 16b, 17, 18, 19a, 19b, 20 and X and Y and had a diploid chromosome number of 2n=50. Of these forms, chromosomes 1a, 1b, 4, 8, 12a/22a, and 12b/22bmay be common derived characters that would link the tree shrew with primates. To define the exact phylogenetic relationships of the tree shrews and the genomic rearrangements that gave rise to the primates and eventually to humans further chromosome painting in Rodentia, Lagomorpha, Dermoptera and Chiroptera is needed, but many of the landmarks of genomic evolution are now known. Received: 11 February 1999; in revised form: 17 June 1999 / Accepted: 20 July 1999     

中国树鼩实验动物化研究进展和展望

该文回顾了我国树鼩驯养繁殖和研究的发展历史,介绍了树鼩实验动物化研究的最新进展,并结合我国目前的状况,提出了今后的工作建议:加强实验树鼩标准化(包括地方和国家标准)的研究、近交系动物的研制、达到商业化树鼩的基础分子与细胞生物学研究工具的研制、人类重大疾病树鼩动物模型研究和建设国家实验树鼩种源基地等。     

实验树鼩肠道螺杆菌属细菌特征分析

近年来,树鼩(Tupaia belangeri)作为一种新型的实验动物被广泛应用于生物医学研究的各个领域。本研究组前期的研究结果显示,螺杆菌属(Helicobacter)是树鼩肠道微生物群落中相对丰度最高的一类细菌,但其具体的细菌种类和结构特征仍然不清楚。因此,本研究将开展实验树鼩肠道中螺杆菌属细菌的分布种类和特征分析,为后续实验研究工作提供资料。通过系统采集72只树鼩的粪便样本,提取核酸后采用巢氏PCR法应用属特异性引物扩增螺杆菌属特异性片段,再分别采用7个种特异性引物对属特异性阳性样本扩增螺杆菌种特异性片段,包括肝螺杆菌(H.hepaticus)、家鼠螺杆菌(H.muridarum)、胆汁螺杆菌(H. bilis)、啮齿类螺杆菌(H. rodentium)、弯曲螺杆菌(Flexispira rappini)、鼩螺杆菌(H. suncus)和盲肠螺杆菌(H. typhlonius)。属特异性引物扩增阳性但种引物扩增阴性的样本进行核酸序列测定和BLAST比对分析,确认其最终所属的螺杆菌种类。结果显示,72份树鼩粪便样本中,属特异性引物扩增阳性有18份,总体阳性率为25.0%。其中,盲肠...     

Repetitive sequences of the tree shrew genome (Mammalia, Scandentia)

Copies of two repetitive elements of the common tree shrew (Tupaia glis) genome were cloned and sequenced. The first element, Tu III, is a ～260 bp long short interspersed element (SINE) with the 5′ end derived from glycine RNA. Tu III carries long polypurine-and polypyrimidine-rich tracts, which may contribute to the specific secondary structure of Tu III RNA. This SINE was also found in the genome of the smooth-tailed tree shrew of another genus (Dendrogale). Tu III appears to be confined to the order Scandentia since it was not found in the DNA of other tested mammals. The second element, Tu-SAT1, is a tandem repeat with a monomer length of 365 bp. Some properties of its nucleotide sequence suggest that Tu-SAT1 is a centromeric satellite.