AMCase、Eotaxin-3 及IL-13 在动物模型中的研究进展

人类疾病的动物模型（Animal Model of Human Diseases）是生物医学科学研究中所建立的动物实验对象和材料。近年来,随 着对动物模型的研究,各种人类疾病的实验模型得到广泛应用。酸性哺乳动物壳多糖酶（AMCase）作为壳多糖酶家族重要成员之 一,与其下游信号分子嗜酸性粒细胞趋化因子（eotaxin-3）以及白细胞介素13（IL-13）的级联免疫反应,近年来成为了动物模型研 究中的热点。本文总结了AMCase、eotaxin-3 及IL-13 在动物模型中的研究进展及其临床意义。     

壳多糖酶研究的概况及最新进展

壳多糖酶专一性水解壳多糖中的β－1,4糖苷键,在自然界的碳循环中具有极其重要的意义．壳多糖酶分布广泛,功能多样,在真菌生长发育、植物抗真菌感染等生理过程中壳多糖酶均发挥重要作用．文章概述了壳多糖酶研究的现状及最新进展,介绍了壳多糖酶的分布、理化性质、催化性质、在细胞中的定位及其调控;简介了近年来真菌和植物壳多糖酶研究的动态及最新进展.     

阿尔茨海默病转基因小鼠的特点和应用

建立动物模型的目的是在实验动物身上复制人类疾病的模型,用于研究人类疾病的病因、发病、病理变化以及疾病的预防和治疗。目前尚无理想的阿尔茨海默病（Alzheimer’s disease,AD）动物模型,AD实验动物模型的滞后在很大程度上制约了AD治疗药物的筛选。随着AD病因和发病机制研究的不断深入,更完善的AD动物模型也在陆续出现。近年来出现的转基因动物模型属于AD的病因模型,但也不能完整复制出AD的所有特征。最大的缺憾在于缺乏神经原纤维缠结（neurofibrillary tangles,NFTs）和在某些转基因模型中（尤其是单转基因模型）无广泛的神经元丢失。虽然用免疫组化方法检测到tau蛋白,但从未发现成对螺旋纤丝（paired helical filaments,PHF）。     

体外红霉素对鼻息肉组织中eotaxin的作用

目的:观察红体外霉素对体外培养鼻息肉组织中嗜酸性粒细胞趋化因子(eotaxin)的作用。方法:从27例住院病人鼻腔中各取鼻息肉标本一份,将每一份均分4份,分对照组、红霉素组培养1d、3d后。每组各取鼻息肉标本经逆转录聚合酶链式反应(RT-PCR)与免疫组织化学染色检测eotaxin表达。结果:①红霉素组中鼻息肉的eotaxinmRNA光度值较对照组低(p<0.05);②两组鼻息肉中eotaxin蛋白灰度值比较,在培养1d后无明显差异,但培养3d后,红霉素组eotaxin蛋白灰度值较对照组低(p<0.05)。结论:体外红霉素能抑制鼻息肉中eotaxin表达,对鼻息肉的临床治疗有指导作用。     

第20届国际生物学奥林匹克竞赛（8）实验考试

实验考试2 生物化学 100分,90min。 介绍： 在酸性条件下,酸性磷酸酶使磷酸化的分子释放磷酸离子。本实验的目的是测定酸性磷酸酶的比活力（性）。任务1,测定土豆粗提物中酸性磷酸酶的活力。任务2,测定任务1粗提物中的蛋白浓度。结合任务1和任务2．可获得酸性磷酸酶的比活力（性）,它的活力用每单位时间每单位蛋白量表示。比活力（性）是蛋白纯度的指标,它的值随蛋白纯度的提高而升高。     

实验动物信息资源网站（2）

（一）国内实验动物信息资源网站 （3）国家遗传工程小鼠资源库 名称：国家遗传工程小鼠资源库 网址：http：／／www．nicemice．org 维护机构：南京大学模式动物研究所 简介：现有小鼠品系264种,在养小鼠达到3万余只。这些小鼠品系中包括糖尿病、肥胖症、白内障、肢体残疾、发育缺陷和心血管系统障碍等多种人类疾病的动物模型。项目组科学家还在研究中发现了一些致病基因。     

刺参酸性粘多糖对诱发性肝癌大鼠的干预作用及免疫功能的影响

目的：通过建立诱发性大鼠肝癌动物模型,研究用刺参酸性粘多糖（SJAMP）对大鼠诱发性肝癌的干预作用及免疫功能的影响。方法：将雄性Wistar大鼠50只随机均分为5个组,正常对照组、模型组和3个SJAMP干预组（A组,B组和c组）,模型组和SJAMP干预组灌胃0．2％DEN生理盐水溶液以诱发肝癌,同时SJAMP干预组按照不同剂量（0．175μg／g,0．35μg／g,O．7μg/g）给予SJAMP,至16周处死大鼠,取血,无菌取脾、胸腺,计算脾指数、胸腺指数。比较各组〉3mm和〉5mm的结节数以及最大结节的体积,计算肿瘤抑制率。贴壁法纯化巨噬细胞,用中性红吞噬实验检测巨噬细胞吞噬功能,MTT法检测巨噬细胞杀伤功能。结果：SJAMP干预组〉3mm和〉5mm的结节数明显少于模型组,最大结节的平均体积明显小于模型组（P〈0．05）;与模型组相比,SJAMP干预组脾指数和胸腺指数明显升高（P〈0．05）,SJAMP干预组巨噬细胞吞噬能力和杀伤功能显著提高（P〈0．05）。结论：刺参酸性粘多糖对大鼠诱发性肝癌有明显的抑制作用;其机制可能是通过刺激免疫器官生长,增强机体的细胞免疫能力来实现的。     

基因编辑动物模型在人类疾病研究中的应用

近年来,随着以CRISPR/Cas9为代表的多种CRISPR系统的开发和不断改进,基因编辑技术逐渐完善,并广泛应用于人类疾病动物模型的制备。基因编辑动物模型为人类疾病的发病机理、病理过程以及预防和治疗等方面的研究提供了重要的素材。目前,用于人类疾病研究的基因编辑动物模型主要有小鼠、大鼠为代表的啮齿类动物模型和以猪为代表的大动物模型。其中啮齿类动物在机体各方面与人类差别较大,且寿命短,无法对人类疾病的研究和治疗提供有效评估和长期追踪;而猪在生理学、解剖学、营养学和遗传学等各方面与人类更接近,是器官移植和人类疾病研究领域重要的动物模型。文中主要介绍了基因编辑动物模型在神经退行性疾病、肥厚心肌病、癌症、免疫缺陷类疾病和代谢性疾病等5种人类疾病研究中的应用情况,以期为人类疾病研究及相关动物模型的制备提供参考。     

靶向肿瘤酸性微环境的抗肿瘤新策略

越来越多的体内体外实验及临床检测都证明了肿瘤酸性微环境对肿瘤的发生、发展和迁移起着重要作用。在肿瘤酸性微环境的重要调节因子中,V型ATP酶（V-ATPases）的作用至关重要。这些蛋白能将离子泵出膜外,在正常细胞和肿瘤细胞中都有着多种功能。本文回顾和总结了该领域的最新研究成果：在异种移植人肿瘤细胞的动物模型中,体内实验显示,采用小分子干扰RNA（siRNA）可抑制V．ATPaseS的功能,加入药物性质子泵抑制剂可显著地抑制人肿瘤细胞的生长。这些结果提示V-ATPases可作为癌症治疗中一个重要的新选择靶标。     

基因敲除小鼠在疾病研究中的应用

人类疾病动物模型在揭示人类疾病的发生机制或建立治疗方法中具有极重要的作用。然而,许多疾病难以用人工诱发的方法制造动物模型,还有许多疾病在实验动物身上不发生,而难以通过自发或人工定向培育的方法获得动物模型。基因敲除技术的出现,为人类精确地研究基因与疾病的直接关系提供了可能,而且可以在个体发生的每个阶段中进行遗传功能的分析。综述了基因敲除小鼠模型在几种疾病研究中的研究与应用及其发展前景。     

Evaluation of AMCase and CHIT-1 expression in monocyte macrophages lineage

Acidic mammalian chitinase (AMCase) and chitotriosidase (CHIT-1) are two active chitinases expressed in humans. The chitinase activity of AMCase was found to be causative in allergic inflammation and its expression was found to be induced by interleukin-13. CHIT1-1 is expressed by phagocytic cells and extremely high levels are seen in lysosomal storage diseases. Despite that AMCase expression in the inflammation is under investigation, little is known regarding its regulation during macrophages' full maturation and polarization. In this study, we compared AMCase and CHIT-1 modulation during monocyte to macrophage transition and polarization. Gene expression analysis was investigated by real-time PCR from mRNA of human monocytes obtained from buffy coat of healthy volunteers, from mRNA of polarized to classically activated macrophages (or M1), obtained by interferon (IFN)-γ and lipopolysaccharide (LPS) treatment, and from mRNA of alternatively activated macrophages (or M2) obtained by interleukin (IL)-4 exposure. Our results showed that the expression of AMCase and CHIT-1 were differently modulated in HMMs at different stage of maturation. The behavior of these two active chitinase suggests that in the immune response their role is complementary.     

Kinetic characterization of recombinant human acidic mammalian chitinase

Human acidic mammalian chitinase (AMCase), a member of the family 18 glycosyl hydrolases, is one of the important proteins involved in Th2-mediated inflammation and has been implicated in asthma and allergic diseases. Inhibition of AMCase results in decreased airway inflammation and airway hyper-responsiveness in a mouse asthma model, suggesting that the AMCase activity is a part of the mechanism of Th2 cytokine-driven inflammatory response in asthma. In this paper, we report the first detailed kinetic characterization of recombinant human AMCase. In contrast with mouse AMCase that has been reported to have a major pH optimum at 2 and a secondary pH optimum around 3-6, human AMCase has only one pH optimum for k(cat)/K(m) between pH 4 and 5. Steady state kinetics shows that human AMCase has "low" intrinsic transglycosidase activity, which leads to the observation of apparent substrate inhibition. This slow transglycosylation may provide a mechanism in vivo for feedback regulation of the chitinase activity of human AMCase. HPLC characterization of cleavage of chitooligosaccharides (4-6-mers) suggests that human AMCase prefers the beta anomer of chitooligosaccharides as substrate. Human AMCase also appears to cleave chitooligosaccharides from the nonreducing end primarily by disaccharide units. Ionic strength modulates the enzymatic activity and substrate cleavage pattern of human AMCase against fluorogenic substrates, chitobiose-4-methylumbelliferyl and chitotriose-4-methylumbelliferyl, and enhances activity against chitooligosaccharides. The physiological implications of these results are discussed.     

Quantification of Chitinase mRNA Levels in Human and Mouse Tissues by Real-Time PCR: Species-Specific Expression of Acidic Mammalian Chitinase in Stomach Tissues

Chitinase hydrolyzes chitin, which is an N-acetyl-D-glucosamine polymer that is present in a wide range of organisms, including insects, parasites and fungi. Although mammals do not contain any endogenous chitin, humans and mice express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Because the level of expression of these chitinases is increased in many inflammatory conditions, including Gaucher disease and mouse models of asthma, both chitinases may play important roles in the pathophysiologies of these and other diseases. We recently established a quantitative PCR system using a single standard DNA and showed that AMCase mRNA is synthesized at extraordinarily high levels in mouse stomach tissues. In this study, we applied this methodology to the quantification of chitinase mRNAs in human tissues and found that both chitinase mRNAs were widely expressed in normal human tissues. Chit1 mRNA was highly expressed in the human lung, whereas AMCase mRNA was not overexpressed in normal human stomach tissues. The levels of these mRNAs in human tissues were significantly lower than the levels of housekeeping genes. Because the AMCase expression levels were quite different between the human and mouse stomach tissues, we developed a quantitative PCR system to compare the mRNA levels between human and mouse tissues using a human-mouse hybrid standard DNA. Our analysis showed that Chit1 mRNA is expressed at similar levels in normal human and mouse lung. In contrast, the AMCase expression level in human stomach was significantly lower than that expression level observed in mouse stomach. These mRNA differences between human and mouse stomach tissues were reflecting differences in the chitinolytic activities and levels of protein expression. Thus, the expression level of the AMCase in the stomach is species-specific.     

Overexpression of chitinase 3-like 1/YKL-40 in lung-specific IL-18-transgenic mice, smokers and COPD

We analyzed the lung mRNA expression profiles of a murine model of COPD developed using a lung-specific IL-18-transgenic mouse. In this transgenic mouse, the expression of 608 genes was found to vary more than 2-fold in comparison with control WT mice, and was clustered into 4 groups. The expression of 140 genes was constitutively increased at all ages, 215 genes increased gradually with aging, 171 genes decreased gradually with aging, and 82 genes decreased temporarily at 9 weeks of age. Interestingly, the levels of mRNA for the chitinase-related genes chitinase 3-like 1 (Chi3l1), Chi3l3, and acidic mammalian chitinase (AMCase) were significantly higher in the lungs of transgenic mice than in control mice. The level of Chi3l1 protein increased significantly with aging in the lungs and sera of IL-18 transgenic, but not WT mice. Previous studies have suggested Chi3l3 and AMCase are IL-13-driven chitinase-like proteins. However, IL-13 gene deletion did not reduce the level of Chi3l1 protein in the lungs of IL-18 transgenic mice. Based on our murine model gene expression data, we analyzed the protein level of YKL-40, the human homolog of Chi3l1, in sera of smokers and COPD patients. Sixteen COPD patients had undergone high resolution computed tomography (HRCT) examination. Emphysema was assessed by using a density mask with a cutoff of -950 Hounsfield units to calculate the low-attenuation area percentage (LAA%). We observed significantly higher serum levels in samples from 28 smokers and 45 COPD patients compared to 30 non-smokers. In COPD patients, there was a significant negative correlation between serum level of YKL-40 and %FEV(1). Moreover, there was a significant positive correlation between the serum levels of YKL-40 and LAA% in COPD patients. Thus our results suggest that chitinase-related genes may play an important role in establishing pulmonary inflammation and emphysematous changes in smokers and COPD patients.     

树鼩在医学实验研究中的新进展

树鼩的许多生物学特性近似于人类,人们利用树鼩成功建立了多种人类病毒感染的动物模型,并在肿瘤、内分泌、神经、视觉等方面有相当广泛和深入的应用和研究。本文概述了树鼩在医学实验研究中的新进展。     

Pulmonary cryptococcosis induces chitinase in the rat

Background

We previously demonstrated that chronic pulmonary infection with Cryptococcus neoformans results in enhanced allergic inflammation and airway hyperreactivity in a rat model. Because the cell wall of C. neoformans consists of chitin, and since acidic mammalian chitinase (AMCase) has recently been implicated as a novel mediator of asthma, we sought to determine whether such infection induces chitinase activity and expression of AMCase in the rat.

Methods

We utilized a previously-established model of chronic C. neoformans pulmonary infection in the rat to analyze the activity, expression and localization of AMCase.

Results

Our studies indicate that intratracheal inoculation of C. neoformans induces chitinase activity within the lung and bronchoalveolar lavage fluid of infected rats. Chitinase activity is also elicited by pulmonary infection with other fungi (e.g. C. albicans), but not by the inoculation of dead organisms. Enhanced chitinase activity reflects increased AMCase expression by airway epithelial cells and alveolar macrophages. Systemic cryptococcosis is not associated with increased pulmonary chitinase activity or AMCase expression.

Conclusion

Our findings indicate a possible link between respiratory fungal infections, including C. neoformans, and asthma through the induction of AMCase.     

Identification of a novel acidic mammalian chitinase distinct from chitotriosidase

Chitinases are ubiquitous chitin-fragmenting hydrolases. Recently we discovered the first human chitinase, named chitotriosidase, that is specifically expressed by phagocytes. We here report the identification, purification, and subsequent cloning of a second mammalian chitinase. This enzyme is characterized by an acidic isoelectric point and therefore named acidic mammalian chitinase (AMCase). In rodents and man the enzyme is relatively abundant in the gastrointestinal tract and is found to a lesser extent in the lung. Like chitotriosidase, AMCase is synthesized as a 50-kDa protein containing a 39-kDa N-terminal catalytic domain, a hinge region, and a C-terminal chitin-binding domain. In contrast to chitotriosidase, the enzyme is extremely acid stable and shows a distinct second pH optimum around pH 2. AMCase is capable of cleaving artificial chitin-like substrates as well as crab shell chitin and chitin as present in the fungal cell wall. Our study has revealed the existence of a chitinolytic enzyme in the gastrointestinal tract and lung that may play a role in digestion and/or defense.