非人灵长类动物基因编辑技术的应用及挑战

建立有效的动物模型是研究人类疾病演进、开发新型治疗手段的重要方法。非人灵长类动物在进化发育、生理生化及病理方面和人类最接近,是研究人类疾病的理想动物模型。随着基因编辑技术的发展,研究者已经成功建立了多种模仿人类疾病的非人灵长类动物模型。但是CRISPR/Cas9的脱靶效应、嵌合突变以及基因敲入效率较低等突出问题也逐渐引起重视。本文综述了基因编辑技术在建立非人灵长类动物模型中的应用现状,提出了目前亟需解决的难点和应对策略,以期为高效、准确构建非人灵长类动物模型提供借鉴与参考。     

恒河猴在生殖生物学中的应用进展

目的非人灵长类动物在生殖生物学研究领域是一种非常重要的实验动物。人类利用非人灵长类动物与人的生物学等方面相似的特征,开展了生殖生物学、生理学、药理学、毒理学以及生育调节等方面的研究工作,为生殖生物学基础研究以及人类健康和疾病问题的基础研究和临床前研究提供了理想的动物模型。随着生命科学的发展,对非人灵长类实验动物质量提出了更高的要求,人们认识到实验时,应用健康的优质非人灵长类动物的重要性。本文简要概括了非人灵长类动物恒河猴的生物学特性,阐述了非人灵长类动物恒河猴在生殖生物学中的应用研究。     

非人灵长类动物精子冻存技术的研究进展

随着生物医学技术的发展,应用非人灵长类动物模型进行基础科学研究日益广泛。与此同时,由于栖息地破坏、狩猎和基因隔离,许多非人灵长类动物濒临灭绝。因此,改进非人灵长类动物精子冻存技术对物种遗传资源的保藏具有重要的意义。本文概述了非人灵长类动物的精液特征,介绍了精液液化和冷冻精子质量评估的方法,分析了冷冻保护剂、冷冻稀释液及冷冻方法等因素对精子冻存效果的影响,总结了目前非人灵长类精子冷冻常用的冷冻保存液和冷冻方法,并对相关精子参数进行了比较,同时探讨了非人灵长类动物精子冻存研究面临的困境,并提出了可行的方案。总之,本文综述了近年来非人灵长类动物精子冻存的重要研究成果,对开发新的冷冻保护剂及改进冷冻技术具有一定的参考价值。     

中华蟾蜍消化道6种酶的组织化学定位

目的研究中华蟾蜍消化道酸性磷酸酶(ACP)、碱性磷酸酶(ALP)、腺苷三磷酸酶(ATPase)、非特异性酯酶(NSE)、过氧化物酶(POX)和琥珀酸脱氢酶(SDH)等6种酶的分布。方法在消化道的8个部位取材,采用冰冻切片技术、石蜡切片技术、酶的组织化学方法和光密度定量分析。结果 ACP主要分布于胃贲门中贲门腺部,十二指肠和回肠中酶反应呈弱阳性。ALP主要分布于食管、十二指肠至回肠的粘膜上皮,十二指肠酶活性最高。ATPase在消化道各部位均有分布,胃中胃腺部和回肠粘膜上皮酶活性显著较高(P0.05)。NSE和POX在整个消化道粘膜上皮和粘膜固有层均有分布,胃各部位酶活性显著较低(P0.05)。SDH除在食管和直肠酶活性显著较低外,其它部位均有大量分布,十二指肠和回肠酶活性显著较高(P0.05)。结论中华蟾蜍消化道粘膜6种酶的分布同其它动物有相似之处,也有其自身特点。6种酶在消化道中的分布与消化道各部位的生理机能密切相关。     

常见消化道黏膜下肿物的临床特点及治疗方法

目的:探讨常见消化道黏膜下肿物(submucosal tumors,SMTs)的临床特点、治疗方式及安全性。方法:选择2014年5月至2016年3月在哈医大一院经内镜切除的并经病理及免疫组化明确诊断的消化道粘膜下肿物的患者共49例。所有患者术后3个月、6个月及12个月后随诊复查胃镜。统计每种肿物的患者临床症状,各种黏膜下肿物的性别分布、大小,及在消化道的分布。计算超声内镜的诊断率,总结病理结果。结果:49例患者中,间质瘤25例,类癌9例,平滑肌瘤8例,异位胰腺4例,脂肪瘤2例,颗粒细胞瘤1例。间质瘤分布以胃底、胃体多见。类癌以直肠最多见,胃内也可见。平滑肌瘤多分布于食管,也可见于胃。异位胰腺多分布于胃窦。通过内镜黏膜下挖除术(Endoscopic submucosal excavation,ESE)切除42例,粘膜下隧道切除术(Submucosal tunneling endoscopic resection,STER)切除4例,还有3例行内镜下黏膜切除(Endoscopic Mucosal Resection,EMR)。所有患者术后均无迟发性出血、严重感染及死亡病例发生。随访3-22个月,所有患者均无复发。结论:黏膜下肿物在内镜及超声内镜各有特点。超声内镜对黏膜下肿物的诊断与治疗具有重要的指导意义。双镜联合使SMTs的内镜下各种治疗方式(EMR、ESE、STER)更有安全保障,使患者受益最大。     

H5N1型禽流感病毒感染非人灵长类动物的观察

目的观察H5N1型禽流感病毒对中国非人灵长类动物的易感性并建立动物模型。方法将病毒通过滴鼻接种实验猴,观察感染后动物的临床症状,采血、咽拭子及各器官组织进行血清学、病原学及病理学检查,记录抗体变化、病毒分离情况及病理学改变。结果感染后动物表现轻度食欲下降、一过性体温升高及外周血白细胞减少,肺组织病毒分离及RT-PCR阳性,病理检查感染急性期动物肺组织表现为间质性肺炎,肺泡间隔增宽,充血出血明显,肺泡受压变形,间质及肺泡内有大量炎细胞浸润,符合病毒性肺炎的改变,感染后14 d动物血清IgG抗体水平较感染前升高4倍。结论H5N1病毒可感染非人灵长类动物,可以作为感染模型进行H5N1病毒的发病机制、疫苗评价、药物筛选等研究。     

非人灵长类肿瘤模型研究进展(英文)

癌症是人类第二大致死的疾病。将体外细胞模型获得的癌症研究结果向临床转化过程中,动物活体实验是必不可少的一个环节。现在的肿瘤活体实验绝大部分采用啮齿类实验动物如小鼠和大鼠,这是因为它们具有个体小、繁殖迅速、遗传背景清楚、转基因技术成熟等优势。但是啮齿类和人的亲缘关系比较远,许多从啮齿类动物模型获得的研究结果不能在人体重现。非人灵长类动物在遗传进化、免疫、生理和代谢等诸多方面与人类高度近似,理论上更加适合癌症研究。本文对现有的非人灵长类肿瘤研究做一综述,主要集中介绍用化学和生物致癌剂在不同的非人灵长类动物诱导肿瘤的研究,为将来用非人灵长类动物研究人类癌症奠定基础。     

非人灵长类胚胎工程研究进展

由于非人灵长类动物与人类在进化上的亲缘关系和生理特征上的高度相似,它们在人类传染病、生殖、发育、代谢、神经和衰老等研究领域具有重要的应用价值。现对非人灵长类动物的常见胚胎工程技术进行综述,包括非人灵长类动物的配子收集、体外受精和胚胎移植等;同时,还结合基因组编辑技术的发展,简要介绍了近年来胚胎工程技术在非人灵长类动物基因编辑方面的应用。     

非人灵长类动物种质冷冻保存研究进展和展望

非人灵长类动物是生物多样性的重要组成部分,也是生物医学研究的珍贵实验动物,然而,由于人类活动、栖息地破坏、狩猎和遗传隔离等原因,许多非人灵长类动物的野生种群数量急剧下降,甚至处于灭绝的边缘。种质冷冻保存对拯救非人灵长类动物和保存遗传物质资源具有重要意义。本文综述了新大陆猴、旧大陆猴和巨猿等类群动物精子、卵子、胚胎和性腺组织等种质冷冻保存的研究进展,介绍了狨猴、松鼠猴、恒河猴、食蟹猴和黑猩猩等种质冷冻保存的主要方法,并对未来种质冷冻保存的研究方向进行了讨论。     

内镜粘膜下剥离术治疗消化道隆起性病变——附58例病例分析

目的:探讨内镜黏膜下剥离术治疗消化道隆起性病变的疗效和安全性。方法:对内镜发现的58例消化道隆起性病变(食管8例,胃26例,十二指肠2例,结肠10例,直肠12例)进行超声内镜检查后(24例位于固有肌层,18例病变位于黏膜下层,4例病变位于黏膜肌层,12例病变位于黏膜层),均进行ESD治疗。结果:48例成功完成ESD治疗,完整切除率达83%,病变最大直径0.5~5.7cm(平均2.9cm),手术时间15~130min(平均74.2min),术中无1例出现皮下气肿,内镜难以控制的大出血,术后未出现迟发性消化道出血,无腹膜炎发热(消化道瘘和胸腔腹腔继发感染)等并发症发生;术中发现2例病变部分突出腔外(底)均转外科手术;2例病变已向深部浸润(胃窦),均转外科手术;术中消化道穿孔6例(胃窦胃底和十二指肠),均转外科急症开腹手术。所有ESD剥离病变包膜完整,基底和切缘未见病变累及。术后随访31例,无1例复发及病变残留。结论:ESD治疗消化道隆起性病变疗效可靠、安全,可以一次性完整地切除病变,提供完整的病理学资料。     

Pediatric hepatic hemangiosarcoma in a rhesus macaque (Macaca mulatta)

Background  Pediatric hepatic angiosarcoma is a rare condition in children with poor prognosis. Microscopically this neoplasm has a particular 'Kaposi-form' arrangement. Hemangiosarcoma in non-human primates is a rare finding.
Methods  Gross and microscopic examination of a 3-year-old rhesus were performed. Immunohistochemistry was used to characterize the hepatic hemangiosarcoma.
Results  The gross necropsy revealed hemoabdomen and a 4 × 3 × 3 cm mass in the liver with multiple smaller masses throughout the hepatic parenchyma. Histopathology confirmed a poorly differentiated hemangiosarcoma. Other organs submitted were free of metastases.
Conclusions  Hemangiosarcoma in non-human primates has been rarely reported. Diagnosis was confirmed by expression of endothelial-specific markers CD31 and vWF by immunohistochemistry. Due to the young age of this monkey and the particular solid pattern throughout the mass this neoplasm resembles pediatric hepatic angiosarcoma in humans.     

Immunohistochemical and morphological features of a small bowel leiomyoma in a black crested macaque (Macaca nigra)

ABSTRACT: BACKGROUND: Spontaneous gastrointestinal neoplasms in non-human primates are commonly seen in aged individuals. Due to genetic similarities between human and non-human primates, scientists have shown increasing interest in terms of comparative oncology studies. CASE PRESENTATION: The present study is related to a case of an intestinal leiomyoma in a black crested macaque (Macaca nigra), kept on captivity by Matecana Zoo, Pereira City, Colombia. The animal had abdominal distension, anorexia, vomiting, diarrhea and behavioral changes. Clinical examination showed an increased volume in the upper right abdominal quadrant caused by a neoplastic mass. The patient died during the surgical procedure. Necropsy revealed several small nodules in the peritoneum with adhesion to different portions of the small and large intestines, liver, stomach and diaphragm. Tissue samples were collected, routinely processed and stained by H&E. Microscopic examination revealed a mesenchymal tumor limited to tunica muscularis, resembling normal smooth muscle cells. Neoplastic cells were positive for alpha-smooth muscle actin and vimentin, and negative for cytokeratin AE1/AE3 by immunohistochemistry. Those morphological and immunohistochemical findings allowed to diagnose the intestinal leiomyoma referred above. CONCLUSION: Neoplastic diseases in primates have multifaceted causes. Their manifestations are understudied, leading to a greater difficulty in detection and measurement of the real impact provides by this disease.     

Changes in thymidine uptake in the gastrointestinal tract of the rat following treatment with 16,16-dimethyl prostaglandin E2

Thymidine uptake in the organs of the gastrointestinal tract of the rat was studied to determine if cell synthesis was involved in the increases in weight of the stomach, small intestine and colon which result from treatment with 16,16-dimethyl prostaglandin E₂ (16,16-dimethyl PGE₂). Animals were treated for 2 days with 16,16-dimethyl PGE₂. They were injected with the ³H-thymidine, sacrificed and the organs of interest were removed. The total amount of tritium in the stomach, duodenum, jejunum, ileum, and colon was determined.Thymidine uptake was significantly increased in the duodenum (1.50 times), jejunum (1.53 times), and colon (1.40 times) but not in the stomach and ileum. The increases were dose related in the duodenum and jejunum. The colon showed a similar dose response pattern but the changes with dose did not reach significance.These results confirm and extend a previous report that 16,16-dimethyl PGE₂ increased thymidine uptake in the duodenum but not the stomach (1). This is different from gastrin which has been shown by others to increase thymidine uptake in the stomach, duodenum, ileum and colon (2,3).     

Brunner’s gland hyperplasia in a geriatric chimpanzee (Pan troglodytes), an infrequently reported lesion

Benign duodenal tumours have very rarely been reported in captive non-human primates and are also rare in human beings. Brunner's gland hyperplasia has not been fully described in a non-human primate. Here, we report Brunner's gland hyperplasia in a geriatric chimpanzee, which was an incidental finding during post-mortem examination.     

Development of a BALB/c mouse model of Helicobacter pylori infection with fresh and frozen bacteria

An experimental model for H. pylori infection was established by intragastrically challenging BALB/c mice with 1 ml (10(8) CFU/ml) of suspension for two consecutive days. Animals were divided into three groups. GA: mice inoculated with fresh bacteria; GB: mice inoculated with frozen bacteria, and GC: mice inoculated with brucella broth (control group). Animals were killed at 7, 14, 21, 28, 35 and 60 days pi and fragments of stomach and duodenum were collected, paraffin embedded and stained by hematoxylin-eosin and Giemsa. The results showed that challenged mice exhibited mild duodenitis and gastritis. In group GA, infiltration in the duodenum was lymphoplasmacytic until day 35; in group GB, it was lymphomonocytic for 60 days pi. In the stomach, H. pylori induced lymphomonocytic infiltration that was present from days 7 to 60 in group GA. In group GB, it was only present from days 14 to 35. In conclusion, our data suggested that freezing altered pathogenic properties of H. pylori and probably inhibited expression of bacterial antigens and consequently the establishment and maintenance of infection. Although the animals developed mild duodenitis and gastritis, the BALB/c mouse is not susceptible to developing peptic ulcers during H. pylori infection.     

Interspecies pair housing of macaques in a research facility

The eighth edition of The Guide for the Care and Use of Laboratory Animals establishes social housing as the 'default' for social species including non-human primates. The advantages of social housing for primates have been well established, but small research facilities housing few primates in indoor cages have struggled with social housing as a result of limitations on appropriate housing and availability of compatible monkeys. Here, we report a novel approach to pair housing macaques - crossing species. We have successfully pair housed an intact male rhesus macaque with an intact male cynomolgus macaque, and an adult female rhesus macaque with numerous subadult female cynomolgus macaques. Monkeys in these pairs established dominant-subordinate relationships similar to same-species pairs. Rhesus and cynomolgus macaques can be successfully paired for the purpose of social housing in facilities with limited numbers of monkeys.     

Comparative study of the transfection efficiency of commonly used viral vectors in rhesus monkey (Macaca mulatta) brains

Viral vector transfection systems are among the simplest of biological agents with the ability to transfer genes into the central nervous system.In brain research,a series of powerful and novel gene editing technologies are based on these systems.Although many viral vectors are used in rodents,their full application has been limited in non-human primates.To identify viral vectors that can stably and effectively express exogenous genes within nonhuman primates,eleven commonly used recombinant adeno-associated viral and lentiviral vectors,each carrying a gene to express green or red fluorescence,were injected into the parietal cortex of four rhesus monkeys.The expression of fluorescent cells was used to quantify transfection efficiency.Histological results revealed that recombinant adeno-associated viral vectors,especially the serotype 2/9 coupled with the cytomegalovirus,human synapsin Ⅰ,or Ca2+/calmodulin-dependent protein kinase Ⅱ promoters,and lentiviral vector coupled with the human ubiquitin C promoter,induced higher expression of fluorescent cells,representing high transfection efficiency.This is the first comparison of transfection efficiencies of different viral vectors carrying different promoters and serotypes in non-human primates (NHPs).These results can be used as an aid to select optimal vectors to transfer exogenous genes into the central nervous system of non-human primates.     

Individual identification of non-human primates using DNA fingerprinting

DNA fingerprints were studied in non-human primates including three species of Old World monkeys and one species of hominoid, using tandem repeats of a 28-base-pair sequence downstream of the human c-Ha-ras-1 oncogene as a probe. We observed Southern hybridization patterns consisting of multiple hypervariable DNA fragments, which were specific to each of the individuals examined. These results indicate that DNA fingerprinting is a powerful tool for identification of individuals among non-human primates, as is the case in man. On leave from the Department of Legal Medicine, Institute of Community Medicine, University of Tsukuba.     

Human is a unique species among primates in terms of telomere length.

TRF (terminal restriction fragments) length in various tissues of non-human primates such as Macaca mulatta (rhesus monkey), Macaca fuscata (Japanese monkey), Macaca fascicularis (crab-eating monkey), Pan troglodytes (common chimpanzee), and Pongo pygmaeus (orangutan) was at least 23 kb without exception, which was quite different from that of human somatic tissues (smaller than 10 kb). The distribution pattern of telomerase activity among tissues was similar between human and non-human primates, while the activity level showed some differences such as that strong telomerase activity was observed in gastrointestinal and lymphocytic tissues from non-human primates. The human appears to be a unique species among primates in terms of telomere length.     

Characterization of the fecal microbiome from non-human wild primates reveals species specific microbial communities

Background

Host-associated microbes comprise an integral part of animal digestive systems and these interactions have a long evolutionary history. It has been hypothesized that the gastrointestinal microbiome of humans and other non-human primates may have played significant roles in host evolution by facilitating a range of dietary adaptations. We have undertaken a comparative sequencing survey of the gastrointestinal microbiomes of several non-human primate species, with the goal of better understanding how these microbiomes relate to the evolution of non-human primate diversity. Here we present a comparative analysis of gastrointestinal microbial communities from three different species of Old World wild monkeys.

Methodology/Principal Findings

We analyzed fecal samples from three different wild non-human primate species (black-and-white colobus [Colubus guereza], red colobus [Piliocolobus tephrosceles], and red-tailed guenon [Cercopithecus ascanius]). Three samples from each species were subjected to small subunit rRNA tag pyrosequencing. Firmicutes comprised the vast majority of the phyla in each sample. Other phyla represented were Bacterioidetes, Proteobacteria, Spirochaetes, Actinobacteria, Verrucomicrobia, Lentisphaerae, Tenericutes, Planctomycetes, Fibrobacateres, and TM7. Bray-Curtis similarity analysis of these microbiomes indicated that microbial community composition within the same primate species are more similar to each other than to those of different primate species. Comparison of fecal microbiota from non-human primates with microbiota of human stool samples obtained in previous studies revealed that the gut microbiota of these primates are distinct and reflect host phylogeny.

Conclusion/Significance

Our analysis provides evidence that the fecal microbiomes of wild primates co-vary with their hosts, and that this is manifested in higher intraspecies similarity among wild primate species, perhaps reflecting species specificity of the microbiome in addition to dietary influences. These results contribute to the limited body of primate microbiome studies and provide a framework for comparative microbiome analysis between human and non-human primates as well as a comparative evolutionary understanding of the human microbiome.