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。     

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...     

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.     

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.     

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。     

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.     

Characterization of 12 polymorphic microsatellite markers in the Chinese tree shrew (Tupaia belangeri chinensis)

The Chinese tree shrew (Tupaia belangeri chinensis) is a small experimental animal with a close affinity to primates. This species has long been proposed to be an alternative experimental animal to primates in biomedical research. Despite decades of study, there is no pure breed for this animal, and the overall genetic diversity of wild tree shrews remains largely unknown. In order to obtain a set of genetic markers for evaluating the genetic diversity of tree shrew wild populations and tracing the lineages in inbreeding populations, we developed 12 polymorphic microsatellite markers from the genomic DNA of the tree shrew. An analysis of a wild population of 117 individuals collected from the suburb of Kunming, China, showed that these loci exhibited a highly expected heterozygosity (0.616). These 12 microsatellites were sufficient for individual identification and parentage analysis. The microsatellite markers developed in this study will be of use in evaluating genetic diversity and lineage tracing for the tree shrew.     

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。     

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.     

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.     

几种冷冻稀释液与单胺类防冻剂对中缅树鼩精子冷冻存活率的影响

该文实验精子采自昆明地区经笼养驯化的树鼩(Tupaia belangeri),检测其冷冻前后运动度、顶体完整率以及检测部分冷冻精子的受精能力。实验一:选用8种已报道的冷冻稀释液TTE、TCG、TCF、TTG、BWW、BTS、DM、SR稀释鲜精,并添加0.4mol/LDMSO,4℃预冷平衡2h后,TTE、DM和SR稀释液的精子的运动度与鲜精无差别(P>0.05),其余处理组均显著下降(P<0.05)。冷冻复苏后,各组的运动度显著低于预冷平衡处理后的运动度(P<0.05);DM组的复苏运动度显著高于其他稀释液组(P<0.05),BWW组最低(P<0.05)。对于顶体完整率,与鲜精相比,4℃平衡2h后,TTE和DM组精子的顶体完整率显著高于BWW、BTS和SR组(P<0.05)。冷冻复苏后,DM组精子的顶体完整率显著高于其它(除了TTE)冷冻组(P<0.05)。实验二:在DM稀释液基础上分别添加4种浓度0.2、0.4、0.8和1.2mol/L的二甲基甲酰胺(DF)、甲酰胺(F)、二甲乙酰胺(DA)和乙酰胺(A)以及0.4mol/LDMSO,经过预冷平衡处理后,与鲜精相比,各防冻剂组的精子运动度没有下降(P>0.05);冷冻解冻后,各冷冻组精子的运动度显著低于预冷平衡处理后的精子运动度(P<0.05);0.8mol/LDF和0.4mol/LDMSO组精子的运动度显著高于其它冷冻组(P<0.05)。对于顶体完整率,预冷平衡处理后各高浓度组的比率显著下降;冷冻复苏后,0.4mol/LF和0.4mol/LDF组精子的顶体完整率相对较高。实验三,人工授精实验中,DM+0.8mol/LDF冷冻精子的受精率为16.7%,DM+0.4mol/LDMSO的受精率为50.0%。以上实验结果提示,含卵黄的非离子冷冻稀释液对树鼩精子冷冻保护效果好,但单胺类防冻剂的防冻效果还需进一步深入研究。     

Cone photoreceptor shedding in the tree shrew (Tupaia belangerii)

Summary Tree shrews were sacrificed at various times during a 12 h light-12 h dark cycle and the retinal pigment epithelium (RPE) was examined for phagosomes. Analysis of photoreceptor densities showed that the tree-shrew retina consists of approximately 96% cone photoreceptors. Therefore, phagosomes in the RPE were assumed to be mostly those of cones. A peak in the number of RPE phagosomes was found about one hour after the onset of light. The number of phagosomes/mm RPE during the light cycle varied from 17.02 at the peak to 2.49 ten hours after light onset. During the dark cycle, values ranged from 0.10 to 0.61 phagosomes/mm RPE. Size profiles of phagosomes showed that large phagosomes peak in number 1/2 h after light onset, while smaller sizes peak at about 1 h after light onset. This may indicate that maximal shedding and phagocytotic activity occurs sometime before the peak in the total number of phagosomes is reached. Statistical corrections for phagosome size, section thickness and phagosomal degradation time were applied to the data in order to assess outer segment renewal time for tree shrew cones.     

Histopathological changes in the liver of tree shrew (Tupaia belangeri chinensis) persistently infected with hepatitis B virus

Herpes simplex virus type-1(HSV-1) and HSV-2 are important human pathogens that cause significant ocular and urogenital complications, respectively. We have previously shown that HSV-1 virions lacking glycoprotein K (gK) are unable to enter into neurons via synaptic axonal membranes and be transported in either retrograde or anterograde manner. Here, we tested the ability of HSV-1 (F) gK-null to protect against lethal challenge with either highly virulent ocular HSV-1 (McKrae strain), or genital HSV-2 (G strain). The gK-null virus vaccine efficiently protected mice against lethal vaginal infection with either HSV-1(McKrae) or HSV-2 (G). Female mice were immunized via a single intramuscular injection with 106 PFU of the gK-null virus. Immunized mice were treated with Depo-Provera fourteen days after vaccination and were challenged via the vaginal route one week later. Ninety percent of mice vaccinated with the gK-null virus survived HSV-1 (McKrae) challenge, while 70% of these mice survived after HSV-2 (G) challenge. Moreover, all vaccinated mice exhibited substantially reduced disease symptoms irrespective of HSV-1 or HSV-2 challenge as compared to the mock vaccinated challenge group. T-cell memory immune responses to specific glycoprotein B (gB) and glycoprotein D (gD) peptide epitopes were detectable at 7 months post vaccination. These results suggest that the highly attenuated, non-neurotropic gK-null virus may be used as an effective vaccine to protect against both virulent HSV-1 and HSV-2 genital infections and induce lasting immune responses.     

树鼩细胞周期蛋白T1 cDNA的克隆和序列分析

树鼩细胞不能感染HIV-1,但支持HIV-1进入靶细胞后的转录,可能是因为树鼩细胞周期蛋白T1(tsCycT1)能结合HIV-1的Tat蛋白。通过设计引物,用RT—PCR技术,获得全长为2175bp tsCycT1基因的cDNA。其核苷酸序列与人的CycT1(hCycT1)基因的cDNA有92．6％的相似性;由此推导出的氨基酸序列有94．1％相似性。其中,hCycT1和tsCycT1氨基端的1—272个氨基酸的相似性高达98．8％,氨基酸第261位点为半胱氨酸。这些结果提示,tsCycT1会和HIV-1的Tat结合,形成高亲和的、锌依赖的复合物,支持HIV-1转录。     

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.     

野生中缅鼩病毒携带情况的初步调查

病毒学检测和监测是树鼩实验动物化和质量控制的重要标准和依据,而野生中缅树鼩是否携带人兽共患病毒鲜见报道。本研究采用酶联免疫吸附(ELISA)方法,对来源于云南昆明市城郊青龙峡地区的野生树鼩是否携带单纯疱疹病毒、轮状病毒、流感病毒、柯萨奇病毒、甲肝病毒、乙肝病毒、丙肝病毒、丁肝病毒、登革热病毒、出血热病毒和麻疹病毒等11种常见病毒进行筛查。结果表明,在已筛查的60只野生中缅树鼩中,可检测到单纯疱疹病毒和柯萨奇病毒,其血清抗体阳性比例分别为36.7%(22/60)和1.67%(1/60),而在粪便中仅检测到轮状病毒,其抗原阳性为6.7%(4/60),未检测到其他病毒,初步显示了野生树鼩自然状态下携带病毒的状况。为此,建议将单纯疱疹病毒、柯萨奇病毒和轮状病毒列为普通级树鼩病毒质量控制的首检项目,进一步大样本筛查将显示是否将其他病毒列为必检项目。     

树鼩CXCR4 cDNA的克隆和序列分析

目的　获得树CXCR4的cDNA序列 ,探讨其是否可以支持HIV_1病毒和细胞的结合。方法　设计相应的引物 ,用RT_PCR ,基因克隆 ,DNA序列分析技术。结果　获得了全长为 10 59bp树CXCR4(tsCXCR4)基因的cDNA。发现其核苷酸序列与人的CXCR4(hCXCR4)基因的cDNA有 92 8%的相似性 ,由此推导出的氨基酸序列有 96 9%相似性。与hCXCR4功能相关的关键位点完全相同 ,tsCXCR4的N端第 7和 12位点为酪氨酸 ,第 14、15和3 2位点为谷氨酸 ,胞外环第 183 ,188为精氨酸 ,第 193、2 62位点以及跨膜区 97位点为天冬氨酸。结论　树的CX CR4很可能会作为HIV_1的辅助受体     

乙型肝炎病毒树鼩体内慢性感染模型的研究历程和展望

乙型肝炎病毒(HBV)感染是全球和我国的重要健康问题,建立方便有效的HBV感染动物体内模型是开展HBV相关基础和临床研究的重要前提。该实验室地处乙型肝炎和肝癌高发流行的广西地区,自建立以来一直以肝癌的病因、发病机制和化学预防作为主要研究方向,于20世纪80年代初开始树鼩体内感染HBV模型方面的研究并持续至今。该文对此研究历程作一概述,并重点介绍关于用新生树鼩慢性感染HBV的新进展。