中缅树鼩微卫星分子标记的筛选

目的 筛选中缅树鼩微卫星分子标记,逐步填补中缅树鼩特异性遗传标记的空白.方法 建立中缅树鼩基因小片段插入文库,利用5’端地高辛标记的(CA)15探针从约1500个菌落中选出36个阳性克隆.对这些克隆进行测序,发现其中15个含有重复序列,其中1个为重复克隆,1个因两端序列太短而不能设计引物.结果 用Primer3软件设计...     

Positively selected genes of the Chinese tree shrew (Tupaia belangeri chinensis) locomotion system

While the recent release of the Chinese tree shrew(Tupaia belangeri chinensis) genome has made the tree shrew an increasingly viable experimental animal model for biomedical research, further study of the genome may facilitate new insights into the applicability of this model. For example, though the tree shrew has a rapid rate of speed and strong jumping ability, there are limited studies on its locomotion ability. In this study we used the available Chinese tree shrew genome information and compared the evolutionary pattern of 407 locomotion system related orthologs among five mammals(human, rhesus monkey, mouse, rat and dog) and the Chinese tree shrew. Our analyses identified 29 genes with significantly high ω(Ka/Ks ratio) values and 48 amino acid sites in 14 genes showed significant evidence of positive selection in the Chinese tree shrew. Some of these positively selected genes, e.g. HOXA6(homeobox A6) and AVP(arginine vasopressin), play important roles in muscle contraction or skeletal morphogenesis. These results provide important clues in understanding the genetic bases of locomotor adaptation in the Chinese tree shrew.     

Establishment of basal cell carcinoma animal model in Chinese tree shrew (Tupaia belangeri chinensis)

Basal cell carcinoma (BCC) is the most common skin cancer worldwide,with incidence rates continuing to increase.Ultraviolet radiation is the major environmental risk factor and dysregulation of the Hedgehog (Hh) signaling pathway has been identified in most BCCs.The treatment of locally advancedand metastatic BBCs is still a challenge and requires a better animal model than the widely used rodents for drug development and testing.Chinese tree shrews (Tupaia belangeri chinensis) are closely related to primates,bearing many physiological and biochemical advantages over rodents for characterizing human diseases.Here,we successfully established a Chinese tree shrew BCC model by infecting tail skins with lentiviral SmoA1,an active form of Smoothened (Smo) used to constitutively activate the Hh signaling pathway.The pathological characteristics were verified by immunohistochemical analysis.Interestingly,BCC progress was greatly enhanced by the combined usage of lentiviral SmoA1 and shRNA targeting Chinese tree shrew p53.This work provides a useful animal model for further BCC studies and future drug discoveries.     

中缅树鼩精子形态特征及冷冻损伤

树鼩作为一种新型的、接近灵长类的实验动物,在医学生物学上的应用受到越来越多的重视。精子的结构特性研究及冷冻后结构的完整性分析是精子生物学的主要内容,也有助于树鼩的实验室快速繁殖。该研究采用人工饲养的中缅树鼩(Tupaia belangeri chinensis),结果显示其睾丸占总体重的(1.05±0.07)%,总体积为(1.12±0.10)mL。附睾尾及输精管精子总量估计在2.2×107～8.8×107,其运动度和顶体完整率分别为(68.8±3.9)%和(90.0±2.1)%。利用扫描电子显微镜和透射电子显微镜对树鼩附睾精子的超微结构进行的观察和分析显示精子头部呈圆形或卵圆形;头部长度、宽度平均分别为6.65和5.82μm;精子尾部中段、主段、尾段和精子总长度平均分别为13.39、52.35、65.74和73.05μm;尾部中段的线粒体螺旋数量为48个,其轴丝结构为典型的"9+9+2"结构。冷冻解冻后的精子主要表现在顶体与质膜不完整、精子断裂、尾部扭曲和膨大。上述结果提示树鼩精子与其他哺乳动物精子的结构特征相似,但是精子大小和线粒体螺旋数目有明显的差别,且超微结构改变仍是冷冻精子运动和受精能力下降的主要原因。     

Evaluating the Phylogenetic Position of Chinese Tree Shrew （Tupaia belangeri chinensis） Based on Complete Mitochondrial Genome： Implication for Using Tree Shrew as an Alternative Experimental Animal to Primates in Biomedical Research

Tree shrew(Tupaia belangeri) is currently placed in Order Scandentia and has a wide distribution in Southeast Asia and Southwest China.Due to its unique characteristics,such as small body size,high brain-to-body mass ratio,short reproductive cycle and life span,and low-cost of maintenance,tree shrew has been proposed to be an alternative experimental animal to primates in biomedical research.However,there are some debates regarding the exact phylogenetic affinity of tree shrew to primates.In this study,we determined the mtDNA entire genomes of three Chinese tree shrews(T.belangeri chinensis) and one Malayan flying lemur(Galeopterus variegatus).Combined with the published data for species in Euarchonta,we intended to discern the phylogenetic relationship among representative species of Dermoptera,Scandentia and Primates.The mtDNA genomes of Chinese tree shrews and Malayan flying lemur shared similar gene organization and structure with those of other mammals.Phylogenetic analysis based on 12 concatenated mitochondrial proteinencoding genes revealed a closer relationship between species of Scandentia and Glires,whereas species of Dermoptera were clustered with Primates.This pattern was consistent with previously reported phylogeny based on mtDNA data,but differed from the one reconstructed on the basis of nuclear genes.Our result suggested that the matrilineal affinity of tree shrew to primates may not be as close as we had thought.The ongoing project for sequencing the entire genome of Chinese tree shrew will provide more information to clarify this important issue.     

Basal physiological parameters in domesticated tree shrews(Tupaia belangeri chinensis)

Establishing non-human primate models of human diseases is an efficient way to narrow the large gap between basic studies and translational medicine. Multifold advantages such as simplicity of breeding, low cost of feeding and facility of operating make the tree shrew an ideal non-human primate model proxy. Additional features like vulnerability to stress and spontaneous diabetic characteristics also indicate that the tree shrew could be a potential new animal model of human diseases. However, basal physiological indexes of tree shrew, especially those related to human disease, have not been systematically reported. Accordingly, we established important basal physiological indexes of domesticated tree shrews including several factors: (1) body weight, (2) core body temperature and rhythm, (3) diet metabolism, (4) locomotor rhythm, (5) electroencephalogram, (6) glycometabolism and (7) serum and urinary hormone level and urinary cortisol rhythm. We compared the physiological parameters of domesticated tree shrew with that of rats and macaques. Results showed that (a) the core body temperature of the tree shrew was 39.59±0.05 °C, which was higher than that of rats and macaques; (b) Compared with wild tree shrews, with two activity peaks, domesticated tree shrews had only one activity peak from 17:30 to 19:30; (c) Compared with rats, tree shrews had poor carbohydrate metabolism ability; and (d) Urinary cortisol rhythm indicated there were two peaks at 8:00 and 17:00 in domesticated tree shrews, which matched activity peaks in wild tree shrews. These results provided basal physiological indexes for domesticated tree shrews and laid an important foundation for diabetes and stress-related disease models established on tree shrews.     

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).     

Recognition memory in tree shrew (Tupaia belangeri) after repeated familiarization sessions

Recognition memories are formed during perceptual experience and allow subsequent recognition of previously encountered objects as well as their distinction from novel objects. As a consequence, novel objects are generally explored longer than familiar objects by many species. This novelty preference has been documented in rodents using the novel object recognition (NOR) test, as well is in primates including humans using preferential looking time paradigms. Here, we examine novelty preference using the NOR task in tree shrew, a small animal species that is considered to be an intermediary between rodents and primates. Our paradigm consisted of three phases: arena familiarization, object familiarization sessions with two identical objects in the arena and finally a test session following a 24-h retention period with a familiar and a novel object in the arena. We employed two different object familiarization durations: one and three sessions on consecutive days. After three object familiarization sessions, tree shrews exhibited robust preference for novel objects on the test day. This was accompanied by significant reduction in familiar object exploration time, occurring largely between the first and second day of object familiarization. By contrast, tree shrews did not show a significant preference for the novel object after a one-session object familiarization. Nonetheless, they spent significantly less time exploring the familiar object on the test day compared to the object familiarization day, indicating that they did maintain a memory trace for the familiar object. Our study revealed different time courses for familiar object habituation and emergence of novelty preference, suggesting that novelty preference is dependent on well-consolidated memory of the competing familiar object. Taken together, our results demonstrate robust novelty preference of tree shrews, in general similarity to previous findings in rodents and primates.     

冷暴露对中缅树鼩适应性产热特征的影响

在冷暴露(5 ± 1℃)1 d,7 d,14 d 和21d 对中缅树鼩的肝脏、心脏、膈肌、腓肠肌和褐色脂肪组织(BAT)的总蛋白和线粒体蛋白含量、线粒体的状态Ⅳ呼吸能力、细胞色素C 氧化酶活力及血清中甲状腺激素的水平等指标进行了测定。结果表明:冷暴露过程中肝脏线粒体蛋白含量对低温的反应比总蛋白的反应剧烈,心脏和BAT 组织的线粒体蛋白含量也随着冷驯化时间的延长而显著增加,但腓肠肌的反应较为温和;各组织线粒体的状态Ⅳ呼吸能力均显著增强,除腓肠肌外其它各组织细胞色素C 氧化酶活性也随冷暴露时间的延长而急剧增加。说明在低温条件下,肝脏、腓肠肌、心脏和BAT 等组织都参与了体温调节的过程,在自然生境中,低温是重要的刺激产热的调节因子。     

Dynamic changes in DNA demethylation in the tree shrew (Tupaia belangeri chinensis) brain during postnatal development and aging

Brain development and aging are associated with alterations in multiple epigenetic systems,including DNA methylation and demethylation patterns.Here,we observed that the levels of the 5-hydroxymethylcytosine (5hmC) ten-eleven translocation (TET) enzyme-mediated active DNA demethylation products were dynamically changed and involved in postnatal brain development and aging in tree shrews (Tupaia belangeri chinensis).The levels of 5hmC in multiple anatomic structures showed a gradual increase throughout postnatal development,whereas a significant decrease in 5hmC was found in several brain regions in aged tree shrews,including in the prefrontal cortex and hippocampus,but not the cerebellum.Active changes in Tet mRNA levels indicated that TET2 and TET3 predominantly contributed to the changes in 5hmC levels.Our findings provide new insight into the dynamic changes in 5hmC levels in tree shrew brains during postnatal development and aging processes.     

Rhombomere-specific patterns of apoptosis in the tree shrew Tupaia belangeri

Whether rhombomere-specific patterns of apoptosis exist in the developing hindbrain of vertebrates is under debate. We have investigated the sequence of apoptotic events in three-dimensionally reconstructed hindbrains of Tupaia belangeri (8- to 19-somite embryos). Apoptotic cells were identified by structural criteria and by applying an in situ tailing technique to visualize DNA fragmentation. Seven rhombomeres originated from three pro-rhombomeres. Among pre-migratory neural crest cells in the dorsal thirds of the neural folds, the earliest apoptotic concentrations appeared in the developing third rhombomere (r3). Dorsal apoptotic maxima then persisted in r3, extended from r3 to r2, and also arose in r5. Transverse apoptotic bands increased the total amount of apoptotic cells in odd-numbered rhombomeres first in r3 and, with a delay, also in r5. This sequence of apoptotic events was paralleled by an approximate rostrocaudal sequence of neural crest cell delamination from the even-numbered rhombomeres. Thus, large-scale apoptosis in r3 and r5 helped to establish crest-free zones that segregated streams of migrating neural crest cells adjacent to r2, r4, and r6. The sequence of apoptotic events observed in the dorsal thirds of rhombomeres matches that reported for the chick embryo. Other shared features are apoptotic peaks in the position of a circumscribed ventricular protrusion of fusing parts of the neural folds in r1 and r2, and Y-shaped apoptotic patterns composed of apoptotic maxima in the dorsal and lateral thirds of r1, r2, and r3. These rhombomere-specific patterns of apoptosis may therefore represent a conserved character, at least in amniotes.This work was supported by the Deutsche Forschungsgemeinschaft (KN 525/1–1, KN 525/1–2, BR 1185/4–1, and former Sonderforschungsbereich 89: Cardiology)     

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。     

Proteome analysis of aflatoxin B1-induced hepatocarcinogenesis in tree shrew (Tupaia belangeri chinensis) and functional identification of candidate protein peroxiredoxin II

In order to explore the proteins responsible for hepatocellular carcinoma (HCC), aflatoxin B(1)-induced hepatocarcinogenesis in tree shrew (Tupaia belangeri chinensis) was analyzed with 2-DE and MS. By comparing HCC samples with their own precancerous biopsies and HCC-surrounding tissues, a group of candidate proteins that differentially expressed in HCC were obtained. Peroxiredoxin (Prx) II, one of the candidates with distinct alteration, was further investigated and validated. Western blot and RT-PCR assays confirmed the overexpression of Prx II in both tree shrew and human HCC tissues. RNA interference for silencing Prx II was employed subsequently to explore the function and underlying mechanism of Prx II on liver cancer cell line Hep3B. Results showed the cell proliferation and clone formation decreased obviously when Prx II expression was inhibited, while the flow cytometer analysis showed the percentage of cell apoptosis enhanced. Inhibition of Prx II expression also obviously increased the generation of ROS and malondialdehyde, both are the products from peroxidation. These results imply the important role of Prx II in hepatocarcinogenesis, possibly through its function in regulating peroxidation and hereby to provide a favorable microenvironment for cancer cell surviving and progressing.     

Eleven novel microsatellite markers for the Chinese alligator (Alligator sinensis)

Chinese alligator (Alligator sinensis) is a critically endangered species endemic to China. In this study, we developed 11 novel microsatellite loci for this rare species and applied them to examine genetic variation of indigenous alligators from Changxing Nature Reserve and America-born Chinese alligators. The 11 polymorphic microsatellites presented a total of 31 alleles among 57 individuals scored, yielding an average of 2.82 alleles per locus. One allele was unique to the American population but four private alleles were detected in the Changxing population. The average expected and observed heterozygosities were 0.400 and 0.482 for the Changxing alligators and 0.520 and 0.621 for the America-born individuals, respectively. These microsatellite markers would be useful tools in the genetic examination of this endangered species.     

Microscopic anatomy of the orbital Harderian gland in the common tree shrew (Tupaia glis)

The orbital Harderian gland of the common tree shrew (Tupaia glis) was investigated at the macroscopic and microscopic levels. In the glands of both sexes only one acinar cell type was found. The cell is characterized by the presence of numerous lipid vacuoles of variable size and by a small number of PAS-positive, electron-dense granules distributed throughout the cytoplasm, which are predominant at the basal portion of each acinar cell. The duct system is well developed within the gland. The content of lipid vacuoles within the acinar cells is secreted from the apical portions by exocytosis, indicating the exocrine function of the organ. Apart from the lipid vacuoles, both acinar and ductal luminal contents of the Harderian gland also contain accretion of electron-dense materials. The vascularization within the Harderian gland is unique in that two capillary types (small fenestrated and irregular sinusoidal capillaries) could be demonstrated. The presence of fenestrated capillaries together with other morphological features (such as accumulation of the small electron-dense granules at the basal pole and the presence of basolateral microvilli) near the basal portion of the acinar cells suggest that the Harderian gland in T. glis might also be involved in an endocrine function.     

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.     

Cryopreservation of epididymal sperm in tree shrews (Tupaia belangeri)

Cryopreservation of sperm from tree shrews, which are considered primitive primates, would enhance genetic management and breeding programs. Epididymal sperm were surgically harvested from male tree shrews, cryopreserved in two Tes-Tris-based cryodiluents, and used in four experiments. In Experiment 1, there were no significant differences in motility and acrosome integrity among five concentrations of egg yolk in TTE after cooling to 4 °C. However, sperm frozen in TTE containing 20% egg yolk at −172 °C/min had better (P < 0.05) post-thaw motility and acrosome integrity. In Experiment 2, sperm held for 10 min prior to storage in liquid nitrogen had greater motility than those held for 5 or 15 min (P < 0.05), but acrosome integrity was not different (P > 0.05) among treatments. In Experiment 3, sperm frozen in TTE diluent had higher (P < 0.05) motility and acrosome integrity than those in TEST diluent. In Experiment 4, there were no differences (P > 0.05) in the fertilization rate of oocytes and the proportion of tree shrews yielding fertilized oocytes, following AI with fresh versus frozen sperm. In conclusion, tree shrew epididymal sperm were successfully cryopreserved, as assessed by post-thaw motility, acrosome integrity, and fertilizing ability.     

树鼩(Tupaia belangeri chinensis)的脊神经丛

由于树鼩在系统发育方面所具的特殊性,以及当今实验动物的小型化趋向,树鼩正在被开发成为新型的实验动物,其生物学特性的研究随之蓬勃开展,如对染色体(陈宜峰等,1981)、神经生理(刘世熠等,1982)、血象(邹如金等,1983)、骨髓象(张耀平等,1986)、血液生化(彭燕章等,1986;陈保生等,1983)、皮纹(Haines,1955;张耀平等,1984)、解剖(Le Gros Clark,1924、1926;George,1977)、     

树鼩(Tupaia belangeri)研究的发展现状和意义

树融分类位置的确定一直存在争议,近年的研究多倾向于将其并入低等灵长类.由于树具有独特的生理结构和功能特点,特别是个体小,繁殖力较弥猴等实验猴类强等特点,从而使它成为一种很有前途的实验动物.