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。     

树鼩的动物福利措施探讨

树鼩在生物学特性、生理生化、新陈代谢和基因组等方面近似于人类,被广泛应用于生理学、病理学、病毒学、免疫学、药理学及遗传学等多个领域。迄今为止用于生物医学研究的树鼩大部分来自野生,实验树鼩尚无国家质量控制标准。为了保证树鼩引种、驯化、饲养、繁殖、质量控制及福利的规范化和科学化,提高树鼩繁殖率和成活率,本文从兽医公共卫生及实验动物科学的角度,对野生树鼩的捕捉、运输、检疫、饲养及繁育等方面的基本原则及技术操作和福利要求作一简述,为从事树鼩工作的有关人员及树鼩实验动物化研究提供参考。     

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。     

树鼩神经肽Y的分子克隆及其灵长类类似物的同源性比较

树鼩由于与灵长类动物有较密切的亲缘关系和其个体小,以及繁殖周期短等特性而倍受关注,尤其是作为医用实验动物的研究,近年来已受到越来越多的重视,但树鼩的分类地位还一直有所争论。该研究从树鼩脑cDNA文库中克隆得到编码树鼩神经肽Y(neuropeptide Y,NPY)前体序列,序列比对发现该序列与灵长类NPY序列同源性高达96.9%。将该序列与GenBank数据库中其他物种的NPY序列构建系统进化树,发现树鼩与灵长类处于同一分支。该研究结果揭示了树鼩与灵长类较近的亲缘关系。     

树鼩进化分类地位的分子证据

树鼩隶属攀鼩目,广泛分布于东南亚、南亚和中国南部等地区。由于其独特的特点,如体型小、脑-体重比例高、生殖周期短、寿命短和饲养成本低等,在生物医学研究中被认为是可望替代灵长类动物的新型实验动物。然而,关于树鼩与灵长类动物的亲缘关系一直存在争议。明确树鼩的分类地位是创建实验动物的重要研究基础。该文介绍了近年来关于树鼩分类地位探讨的分子证据。在现有的研究中,大部分核DNA序列研究,包括近期树鼩全基因组序列分析,都支持树鼩是灵长动物的近缘旁系群,然而绝大部分基于线粒体DNA序列的研究却显示树鼩与啮齿动物的亲缘关系更为接近。这样的分歧主要是由于线粒体序列和核基因数据的差异以及不同的算法导致。综合现有不同DNA数据的研究结果,作者认为树鼩作为灵长类的近亲这一结论应该成为共识。     

实验树鼩配合饲料的研究与应用

根据树鼩的生物学特性并参考有关实验动物饲料标准为树鼩设计和加工生产一种实验饲育饲料。结果表明该饲料能满足树鼩的健康生长、饲养繁殖和实验树鼩的营养需求。该饲料的营养成分和混合均匀度均符合地方标准,生产和饲喂方便,具有一定的应用和推广前景。     

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。     

树鼩驯养繁殖实验动物标准及丙型肝炎动物模型技术平台建设的探讨

目前用于生物医学研究的树鼩大多数来源于野生捕获,由于个体间的差异较大,其实验结果的均匀性和可重复性较差,亟待解决树鼩人工饲养设施条件、繁殖关键技术、动物质量标准等一系列基础科学问题。许多文献报道了树鼩与其他实验动物比较,在肝病研究中具有巨大的开发应用潜力,在丙型肝炎动物模型研究中,亟待解决丙型肝炎病毒(HCV)细胞培养体系、HCV病毒感染剂量与感染途径、HCV病毒对树鼩体外、体内感染后病毒核酸检测、抗体检测和病理检测等技术方法的建立及动物模型评价标准问题。本文就上述问题系统性地进行初步报道,旨在为树鼩早日实现实验动物化和丙型肝炎疾病研究中应用提供科学依据。     

综述与专论: 树鼩病毒感染模型的研究新进展

病毒感染引起的疾病接近中国主要传染病发病率的一半,也是传染病致死的主要病因。建立与人类亲缘关系较近、方便有效的感染人类病毒的动物模型,对了解病毒的生物学特性、感染致病机理及制定有效防控措施具有重要意义。树鼩作为灵长类动物的近亲,与人类在生理生化、基因组学等方面的相似性远高于大鼠、小鼠等常用啮齿类实验动物,并具有个体小、便于实验操作、饲养成本低、能感染多种人类病毒等特点,作为动物模型在病毒感染性疾病研究中突显优势和潜能。本文从地区分布、进化、生物学特性等方面,阐述了树鼩作为动物模型应用于病毒感染性疾病研究的优势,包括在乙型肝炎病毒、甲型肝炎病毒,及其他病毒感染疾病研究中的进展。     

树鼩研究综述

树鼩是一种小型哺乳类,其皮张可以利用,特别是近代用于医学实验动物日益增多,对其研究亦相应增多,现将国内外有关研究作一综述供有关方面参考。1 分类树鼩(Tupaia)属攀鼩目(SCAN NDETIA)树鼩科(Tupaiidae),全世界有11种,主要     

树鼩模型:抑郁症的社会竞争失败与学习和记忆的被捕获条件反射

最近基因组研究表明树鼩属于灵长类或是与灵长类亲缘关系最密切的姐妹种.因此,树鼩可能是应用于建立人类疾病动物模型的最佳动物之一.该文报道一种抑郁症的社会竞争失败病因学树鼩(Tupaia belangerichinensis)模型.一对雄性树鼩被饲养在一个双笼中,用网格把双笼隔开,网格上有一小门.适应1周后,把小门打开,这一对树鼩产生短暂的争斗,每天一次,连续21天.其中争斗失败者被称为服从者.这个过程可导致每天1 h的直接社交冲突和23 h的间接相互影响(比如通过气味、视觉等).与正常对照相比,失败者在第三周也就是社交冲突的最后一周显示了体重、自主活动、躲避行为、尿液皮质醇水平等的变化,并且这种改变可持续至少2周以上.此外,还报道全新的记忆模型,一种被捕获条件反射树鼩模型.在一个封闭的小房间中放置捕获笼,其中挂有一片苹果,小房间中有一只自由活动的树鼩.训练的前4次树鼩进入捕获笼吃苹果并不触发捕获笼关闭,但在第5次时触发捕获笼关闭,并持续一小时才释放树鼩.第1-5次树鼩进入捕获笼的延迟时时间作为适心性指标,其中第5次才是作为被捕获的一次学习训练.24 h后,测试树鼩进入捕获笼的延迟时间作为被捕获记忆能力指标.树鼩经过第5次被捕获训练,能形成很好的被捕获记忆,因为24 h后的延迟时间极人地增加.在训练前腹腔注射已知能阻断记忆形成的NMDA受体拮抗剂MK-801(0.2 mg/kg,腹腔注射),对适应指标没有显著影响,但足极大地缩短了24 h后测试的延迟时间,即阻断了被捕获记忆.这些结果表明了一种抑郁症的慢性社会竞争失败与学习和记忆的一次被捕获条件反射树鼩模型.这两种树趵模型对抑郁症与学习和记忆的机理研究、抗抑郁症新药的临床前药效学评价具有潜在的重要意义.

Abstract：

Recent genome studies indicate that tree shrew is in the order or a closest sister of primates, and thus may be one of the best animals to model human diseases. In this paper, we report on a social defeat model of depression in tree shrew (Tupaia belangeri chinensis). Two male tree shrews were housed in a pair-cage consisting of two independent cages separated by a wire mesh partition with a door connecting the two cages. After one week adaptation, the connecting door was opened and a brief fighting occurs between the two male tree shrews and this social conflict session consisted of 1 h direct conflict (fighting) and 23 h indirect influence (e.g. smell, visual cues) per day for 21 days. The defeated tree shrew was considered the subordinate. Compared with naive animals, subordinate tree shrews at the final week of social conflict session showed alterations in body weight, locomotion, avoidance behavior and urinary cortisol levels.Remarkably, these alterations persisted for over two weeks. We also report on a novel captive conditioning model of learning and memory in tree shrew. An automatic trapping cage was placed in a small closed room with a freely-moving tree shrew. For the first four trials, the tree shrew was not trapped when it entered the cage and ate the bait apple, but it was trapped and kept in the cage for 1 h on the fifth trial. Latency was defined as the time between release of the tree shrew and when it entered the captive cage. Latencies during the five trials indicated adaptation. A test trial 24 h later was used to measure whether the one-trial trapping during the fifth trial could form captive memory. Tree shrews showed much longer trapping latencies in the test trial than the adaptation trials. The N-methyl-d-aspartate (NMDA) receptor antagonist MK-801 (0.2 mg/kg, i.p.), known to prevent the formation of memory, did not affect latencies in the adaptation trails, but did block captive memory as it led to much shorter trapping latencies compared to saline treatment in the test trial. These results demonstrate a chronic social defeat model of depression and a novel one-trial captive conditioning model for learning and memory in tree shrews, which are important for mechanism studies of depression, learning,memory, and preclinical evaluation for new antidepressants.     

滇西亚种树鼩体外寄生虫自然感染状况及分析

目的调查滇西亚种树鼩体外寄生虫的自然感染状况,为建立树鼩质量控制标准提供依据。方法对野生的滇西亚种树鼩和自繁F1代树鼩各60只分别用尸检法和活体法检测,随即采用拔毛或用透明胶带粘取腋下、耳根、腹股沟、肛门附近等处的毛发制片,置于体视镜和显微镜下观察虫体和虫卵。结果野生滇西亚种树鼩和自繁F1代树鼩体外寄生虫自然感染率分别为：虱子25%/6%、蚤5%/0、蜱1.6%/0、水蛭1.6%/0、虱子和蚤混合感染1.6%/0。结论野生树鼩体外寄生虫的自然感染多为虱子和蚤,其次还有蝉、水蛭,总感染率明显高于自繁F1代。使用灭虱灵对于驱除树鼩体外寄生虫具有较好的疗效。     

Motor Function in MPTP-Treated Tree Shrews (Tupaia belangeri chinensis)

The tree shrew, a new experimental animal model, has been used to study a variety of diseases, especially diseases of the nervous system. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is the gold standard for toxin-based animal models of Parkinson’s disease (PD) because MPTP treatment replicates almost all of the pathological hallmarks of PD. Therefore, in this study, the effects of MPTP on the motor function of the tree shrew were examined. After five daily injections of a 3 mg/kg dose of MPTP, the motor function of MPTP-injected tree shrews decreased significantly, and the classic Parkinsonian symptoms of action and resting tremor, bradykinesia, posture abnormalities, and gait instability were observed in most MPTP-injected tree shrews. HPLC results also showed significantly reduced striatal dopamine and 3,4-dihydroxyphenylacetic acid levels in tree shrews after MPTP injection. Increased oxidative stress levels are usually considered to be the cause of dopaminergic neuron depletion in the presence of MPTP and were observed in the substantia nigra of MPTP-treated tree shrews, as indicated by a significant reduction in superoxide dismutase and glutathione peroxidase activity and increased levels of malondialdehyde. In addition, elevated α-synuclein mRNA levels in the midbrain of MPTP-treated tree shrews were observed. Furthermore, MPTP-treated tree shrews showed the classic Parkinsonian symptoms at a lower MPTP dosage compared with other animal models. Thus, the MPTP-treated tree shrew may be a potential animal model for studying the pathogenesis of PD.     

驯养中缅树鼩的消化道寄生虫监测及寄生蠕虫驱虫效果

目的了解野外树鼩和驯养树鼩寄生虫感染情况及其驱虫效果。方法 随机选取野外捕获3 d和已驯1个月(未驱虫)、F1代、F2代、F3代树鼩,各五笼,每笼3～4只,用饱和食盐水漂浮法检查粪样虫卵或卵囊,根据形态学鉴定虫种。结果粪样共检获两种寄生性线虫虫卵(毛尾线虫和奇口线虫)、膜壳绦虫卵、一种球虫卵囊。野外捕获3 d和驯养1个月树鼩的线虫卵检出率最高,达100%;绦虫卵检出率分别达60%和20%;球虫卵囊检出率分别达40%和20%。驯养F1代、F2代、F3代树鼩,仅F2代1笼检出线虫卵,检出率为20%。驱虫当天粪样检出的线虫虫卵、绦虫卵和球虫卵囊与驱虫前检出结果比例一致,并收集到39条奇口线虫、3条毛尾线虫、18条长膜壳绦虫。驱虫后第2天和第3天均未收集到虫体,粪样中也未检测到线虫卵和绦虫卵,仅检测到球虫卵囊。结论为树鼩实验动物化的消化道寄生虫病的监测和控制提供基础信息。     

树[鼠句]的基本实验操作方法

目的对树[鼠句]抓取和保定、采血、灌胃基本实验技术方法进行探讨,逐步规范树[鼠句]实验技术。方法选用成年树[鼠句]进行抓取和保定,分徒于法和器具法（自制捕捉保定袋）,对130只树[鼠句]采用尾静脉、股动（静）脉两种采血方法;采取人用8号胃管可对树[鼠句]绎口灌胃给药。结果所采用徒手、器具的方法抓取和保定树[鼠句]均能有效地控制动物,不会发生动物死亡或很少逃逸;两种方法都顺利采集到所需血量,股动（静）脉单次最大采血量可达2mL而不损伤动物;12只树[鼠句]连续灌胃10d,成功率100％。结论自制的捕捉保定袋经济实用,摸索的几种树[鼠句]实验技术方法具有操作简便、安全、快捷等优点。     

Sequence diversity of the MHC Ⅱ DRB gene in Chinese tree shrews (Tupaia belangeri chinensis)

The major histocompatibility complex (MHC) is a cluster of genes involved in vertebrate immune response regulation. MHC class I and II cell surface proteins are crucial for discrimination of self versus non-self by the adaptive immune system. Due to their special phylogenetic position within the Euarchontoglires and as a relative of primates, tree shrews have been proposed as an alternative experimental animal model for biomedical studies. However, information about the genetic structure of the tree shrew populations is largely unknown. In this study, we characterized diversity in exon 2 of the MHC II DRB gene isolated from Chinese tree shrews (Tupaia belangeri chinensis). We identified 12 different DRB exon 2 alleles from 15 Chinese tree shrews, 1 to 4 alleles were observed per individual with high levels of sequence divergence between alleles. There were more non-synonymous than synonymous substitutions in the functionally important antigen-binding site (dN/dS = 2.7952, P < 0.01), indicating that the DRB exon 2 in Chinese tree shrews has been influenced by positive selection.     

生活环境条件对中国树鼩血激素水平和心理行为的影响

目的探索生活环境条件(居住环境条件和动物之间相互作用的因素)对中国树鼩血激素水平和心理行为的影响。方法采用空间大小不等的笼具饲养中国树鼩,或给予利血平,分别在15、30、60、120、180 d时用乙醚吸入麻醉,从心脏采血,用放射免疫法检测血液中睾酮(testosterone,T)、雌二醇(estradiol,E2)、内皮素(endothelin,ET)、肾上腺素(adrenaline,A)和去甲肾上腺素(noradrenaline,NA)水平。结果①将中国树鼩分别放入大笼(D1组)、小笼(X1组)单独饲养15、30、60、120、180 d时检测,与大笼(D1组)比:小笼(X1组)T水平显著降低(P0.01),A、NA、ET水平显著升高(P0.01)。②小笼和大笼临近饲养(X2组)饲养15、30、60、120、180 d时检测,X2组T、A、NA水平均比小笼单独饲养(X1组)显著升高(均P0.01)。③利血平各组A、NA水平均显著降低(均P0.01)。④小笼单独饲养(X1组)、小笼和大笼临近饲养(X2组)的动物均出现猝丧、食欲降低、睾丸萎缩、阴茎外露下垂等应激症状。利血平组中国树鼩均出现性情温顺,活动显著减少,食欲降低,停喂利血平,放入大笼饲养,动物逐渐恢复到正常生活状态。结论居住环境条件和动物之间的相互作用均能引起中国树鼩血激素水平变化和心理行为的改变。     

野生和人工繁育树鼩部分生理指标检测与比较

比较野生和人工繁育树鼩的部分生理指标,为人类疾病的动物模型创制提供基本参数.血液采自昆明地区54只野生树鼩(被捕获后人工饲养1-2月)和54只子一代人工繁育树鼩.该研究首次报道在两组动物中,肌酸激酶、肌钙蛋白I、总胆汁酸、果糖胺、低密度脂蛋白胆固醇等在性别间差异无显著性;这些指标在人工繁育树鼩中的中位数(四分位数间距)依次为:1449(956)U/L、5.94(7.23)μg/L、15.6(19.7)μmol/L、393.5(80.8)μmol/L和0.36(0.32)mmol/L;在野生树鼩中依次为:986(564)U/L、4.01(4.10)μg/L、20.0(20.6)μmol/L、379.0(104.0)μmol/L和0.46(0.23)mmol/L.人工繁育树鼩生理指标表现出个体间离散程度降低趋势,但个别反映肝脏功能及心肌情况的指标表现出平均值上升,个体离散程度扩大的现象.这些生理指标为人类疾病的树鼢模型创制打下了基础.     

Characterization of tree shrew telomeres and telomerase

The use of tree shrews as experimental animals for biomedical research is a new practice. Several recent studies suggest that tree shrews are suitable for studying cancers, including breast cancer, glioblastoma,lung cancer, and hepatocellular carcinoma. However, the telomeres and the telomerase of tree shrews have not been studied to date. Here, we characterize telomeres and telomerase in tree shrews. The telomere length of tree shrews is approximately 23 kb, which is longer than that of primates and shorter than that of mice, and it is extended in breast tumor tissues according to Southern blot and flow-fluorescence in situ hybridization(FISH) analyses. Tree shrew spleen, bone marrow, testis, ovary, and uterus show high telomerase activities, which are increased in breast tumor tissues by telomeric repeat amplification protocol assays. The telomere length becomes shorter, and telomerase activity decreases with age. The tree shrew TERT and TERC are more highly similar to primates than to rodents. These findings lay a solid foundation for using tree shrews to study aging and cancers.