《细胞研究》：世界首个PPARγ基因敲除猪诞生

中国科学院广州生物医药与健康研究院赖良学教授领导的研究团队与美国密歇根大学心血管研究中心陈育庆教授领导的研究团队合作,首次将锌指核酸酶基因打靶技术应用于猪内源性基因敲除研究,成功敲除了猪内源性PPARγ基因。此研究在世界上首次建立了对糖尿病和心血管并发症的研究有重要应用价值的PPARγ基因敲除猪模型。研究成果于2011年4月19日在线发表于国际著名期刊《细胞研究》（Cell Research）上。目前,敲除大动物的内源性基因只有通过体细胞基因敲除结合克隆技术才能够实现。由于体细胞基因敲除效率极低,     

代谢病相关的遗传工程小鼠模型

糖尿病及肥胖症等代谢性疾病已成为影响人类健康的主要疾病,属于多基因所致的代谢综合征,遗传模式复杂多样,至今仍所知甚少。理想的实验动物模型是我们深入了解代谢病病因、遗传及环境因素的必要工具,并且可以用来研究验证新的治疗药物。近年来,已经获得了大量的遗传工程动物模型,包括转基因、基因敲除模型等遗传工程动物,对于代谢性疾病的研究意义重大。本文主要介绍近年来应用较多的糖尿病及肥胖相关的遗传工程小鼠模型遗传特征及应用。     

GPR126条件性基因敲除载体的快速构建

基因敲除小鼠模型是在动物体内研究基因功能的重要和可靠的手段之一,而构建用于同源重组的基因敲除载体是构建基因敲除小鼠模型的关键一步.条件性基因敲除技术将针对靶基因的修饰作用限制在小鼠的特定发育时期或特定类型的组织细胞中,通过加入具有位点特异性的重组系统,该修饰作用在时间和空间上均处于可调控的状态;条件性基因敲除技术既可以避免某些具有重要功能基因的敲除所带来的早期胚胎致死问题,还可用于精确分析某些多效基因在不同组织或不同发育阶段的特定功能差异.通过合理利用改良的Red重组系统,将两个LoxP位点快速准确地插入到了目标位置,实现了GPR126条件性基因敲除载体的快速构建,为后续的构建GPR126基因敲除小鼠模型、在动物体内研究基因的功能奠定了基础.     

SHBG基因敲除小鼠模型的建立及其表型分析

目的:建立性激素结合球蛋白(SHBG)基因条件敲除小鼠模型,为探讨胎盘组织中SHBG在体内的生理功能及其与妊娠期糖尿病发病关系提供实验手段。方法:首先运用生物信息学手段确定小鼠SHBG基因组序列,构建SHBG打靶载体,以电穿孔方法将其导入小鼠ES细胞,筛选培养阳性ES细胞并行PCR鉴定,并将正确同源重组的ES细胞注射进小鼠囊胚,移入受体小鼠子宫;将获得的嵌合体小鼠与C57BL/6J小鼠交配,筛选后获得Flox小鼠,该小鼠与EIIa-Cre转基因小鼠杂交,子代多次自交获得SHBG全身基因敲除(SHBG~(-/-))的小鼠。结果:运用同源重组及ES细胞技术建立了SHBG基因的Flox小鼠,并利用Cre/Loxp重组酶系统建立了SHBG基因全身敲除小鼠模型,PCR方法从基因水平证明了SHBG基因Flox小鼠及SHBG基因全身敲除小鼠模型建立成功。对基因敲除鼠进行初步表型分析发现:SHBG基因全身敲除小鼠的生长发育与野生型小鼠相比无明显肉眼所见异常,SHBG基因全身敲除雌雄小鼠均具有生殖能力。结论:成功建立SHBG基因全身敲除小鼠模型,通过对基因敲除鼠进行初步表型分析,发现SHBG基因全身敲除小鼠外观上发育正常,为进一步研究SHBG在妊娠期糖尿病中的作用奠定了基础。     

基因敲除动物的研究和应用

目的基因敲除动物是近十几年来发展起来的在个体水平上研究基因功能的一类实验动物,它以基因敲除技术和胚胎干细胞技术为基础,在生命科学研究的各个领域得到了广泛应用。最近两年发展起来的RNA干扰技术仍然不能代替它。本文综述了基因敲除动物在各医学生物领域的研究与应用、浅谈其与RNA干扰技术的比较及其发展前景。     

CRISPR-Cas介导的基因编辑工具

近年来,随着人们对CRISPR-Cas系统研究的不断深入和改造,CRISPR-Cas系统已成为一项新的基因靶向修饰技术,能够定向对靶基因进行定向沉默,目前该项技术已经成功地用于HEK293、小鼠及斑马鱼的细胞并产生了稳定的基因敲除细胞株,在小鼠、果蝇等模式动物上成功构建了基因敲除模型。CRISPR-Cas以其设计简单,耗时短、效率高等优点使其成为一项具有广阔应用前景的基因靶向敲除技术。就CRISPR-Cas的发现、结构、作用机理以及Cas9/gRNA目前的一些应用进行综述。     

高脂饮食联合链脲佐菌素建立2型糖尿病大鼠模型的研究进展

2型糖尿病大鼠模型的建立能够为糖尿病及其并发症的发病机制、预防、诊断及治疗的研究提供理想的动物实验技术平台。2型糖尿病大鼠模型的成模率受多种因素的影响,本文就建立2型糖尿病大鼠模型的主要影响因素STZ的应用、高脂高糖饮食及大鼠的选择与饲养等做一综述。以期对建立2型糖尿病大鼠模型的方法提供一定的参考价值。     

研发动态

世界首例利用锌指核酸酶技术培育的内源性基因敲除猪诞生中国科学院广州生物医药与健康研究院的研究团队与美国密歇根大学心血管研究中心合作,首次将锌指核酸酶基因打靶技术应用于猪内源性基因敲除研究,成功敲除了猪内源性PPARγ基因。这是世界上首次建立PPARγ基因敲除猪模型,对糖尿病和     

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。     

基于人工核酸酶的遗传修饰技术在线虫中的应用

近年来,基于人工核酸酶TALEN或CRISPR-Cas9的遗传编辑技术表现出高效、快捷和靶向性修饰等特点,已迅速成为生物学研究中的重要手段。在经典的遗传学模式动物线虫中,这两项技术的应用主要包括:生殖腺基因敲除、条件性基因敲除、定点突变以及单拷贝基因插入等。将介绍这方面研究进展,并展望这两项技术在线虫遗传学研究中的应用前景。     

A critical review of mathematical models and data used in diabetology

The literature dealing with mathematical modelling for diabetes is abundant. During the last decades, a variety of models have been devoted to different aspects of diabetes, including glucose and insulin dynamics, management and complications prevention, cost and cost-effectiveness of strategies and epidemiology of diabetes in general. Several reviews are published regularly on mathematical models used for specific aspects of diabetes. In the present paper we propose a global overview of mathematical models dealing with many aspects of diabetes and using various tools. The review includes, side by side, models which are simple and/or comprehensive; deterministic and/or stochastic; continuous and/or discrete; using ordinary differential equations, partial differential equations, optimal control theory, integral equations, matrix analysis and computer algorithms.     

Mouse models created to study the pathophysiology of Type 2 diabetes

Obesity and Type 2 diabetes have become epidemics in the Western world. Understanding the pathophysiology of the disease should help in prevention and treatment of these disorders. A common theme is the presence of insulin resistance that eventually results in Type 2 diabetes. To understand the underlying mechanisms in the progression of the disease states, investigators have created mouse models by transgenic overexpression of a candidate gene or produced gene-deletion mouse models. This review will summarize many of the more appropriate models that study insulin resistance and Type 2 diabetes.     

非人灵长类糖尿病动物模型研究进展

糖尿病是继心血管疾病和肿瘤之后的另一种严重危害人类健康的重要慢性疾病,据世界卫生组织(WHO)报道,2009年全世界约有2.2亿糖尿病患者。对糖尿病发病机理的研究、预防和诊断、治疗药物的筛选和评价都需要合适的动物模型。在已报道的糖尿病动物模型中,非人灵长类动物糖尿病病程、病症与人类的糖尿病最为相似。该文从糖尿病动物模型的来源归纳了目前报道的主要的非人灵长类糖尿病模型,重点介绍了猕猴、食蟹猴和树鼩糖尿病模型及其特征,并对该领域的发展提出了一些思考。     

Pharmacokinetics of oltipraz in rat models of diabetes mellitus induced by alloxan or streptozotocin

Pharmacokinetic parameters of oltipraz were compared after intravenous (10 mg/kg) and oral (30 mg/kg) administration in rat model of diabetes mellitus induced by alloxan (rat model of DMIA) or streptozotocin (rat model of DMIS) and their respective control male Sprague-Dawley rats. In rat models of DMIA and DMIS, the expressions and mRNA levels of CYP1A2, 2B1/2, and 3A1(23) increased, and oltipraz was metabolized mainly via CYP1A1/2, 2B1/2, 2C11, 2D1, and 3A1/2 in male Sprague-Dawley rats. Hence, it would be expected that the AUC and CL values of oltipraz would be significantly smaller and faster, respectively, in rat models of diabetes. This was proven by the following results. After intravenous administration, the AUC values were significantly smaller in rat models of DMIA (40.1% decrease) and DMIS (26.0% decrease) than those in respective control rats, and this could be due to significantly faster CL values in rat models of DMIA (40.1% increase) and DMIS (26.0% increase). The faster CL could be due to increase in hepatic blood flow rate and significantly faster CL(int) in rat models of diabetes, since oltipraz is an intermediate hepatic extraction ratio drug in male Sprague-Dawley rats. After oral administration, the AUC values of oltipraz were also significantly smaller in rat models of DMIA (54.0% decrease) and DMIS (63.2% decrease). This could be due to increase in hepatic blood flow rate, significantly faster CL(int), and changes in the intestinal first-pass effect in rat models of diabetes. However, this was not due to decrease in absorption in rat models of diabetes.     

Islet Remodeling in Female Mice with Spontaneous Autoimmune and Streptozotocin-Induced Diabetes

Islet alpha- and delta-cells are spared autoimmune destruction directed at beta-cells in type 1 diabetes resulting in an apparent increase of non-beta endocrine cells in the islet core. We determined how islet remodeling in autoimmune diabetes compares to streptozotocin (STZ)-induced diabetes. Islet cell mass, proliferation, and immune cell infiltration in pancreas sections from diabetic NOD mice and mice with STZ-induced diabetes was assessed using quantitative image analysis. Serial sections were stained for various beta-cell markers and Ngn3, typically restricted to embryonic tissue, was only upregulated in diabetic NOD mouse islets. Serum levels of insulin, glucagon and GLP-1 were measured to compare hormone levels with respect to disease state. Total pancreatic alpha-cell mass did not change as autoimmune diabetes developed in NOD mice despite the proportion of islet area comprised of alpha- and delta-cells increased. By contrast, alpha- and delta-cell mass was increased in mice with STZ-induced diabetes. Serum levels of glucagon reflected these changes in alpha-cell mass: glucagon levels remained constant in NOD mice over time but increased significantly in STZ-induced diabetes. Increased serum GLP-1 levels were found in both models of diabetes, likely due to alpha-cell expression of prohormone convertase 1/3. Alpha- or delta-cell mass in STZ-diabetic mice did not normalize by replacement of insulin via osmotic mini-pumps or islet transplantation. Hence, the inflammatory milieu in NOD mouse islets may restrict alpha-cell expansion highlighting important differences between these two diabetes models and raising the possibility that increased alpha-cell mass might contribute to the hyperglycemia observed in the STZ model.     

诱发性2型糖尿病小鼠模型与自发性db/db小鼠特性的比较

目的建立诱发性2型糖尿病小鼠模型,并将其与自发性2型糖尿病小鼠db/db进行比较分析。客观评价两种2型糖尿病小鼠模型,为糖尿病研究中动物模型的选择与实际应用提供实验依据。方法高脂饲料喂养C57BL/6J小鼠4周,腹腔连续3次注射STZ,建立诱发性2型糖尿病小鼠模型。感染后4周,大体肉眼观察小鼠的肝脏、肾脏,测定糖耐量,血清生化指标及血清细胞因子IL-2、IL-4、IL-6、IFN-γ、TNF-α、IL-17、IL-10表达量,将其与同龄的自发性2型糖尿病小鼠db/db进行比较分析。结果肉眼观察发现,两组模型小鼠的肝脏、肾脏与对照组均具有明显差异。糖耐量分析中,两组模型小鼠与对照组小鼠各时间点的血糖值均具有统计学差异（P〈0.05）,耐糖功能低下,两组模型小鼠间血糖值无统计学差异。血液生化指标中,与对照组小鼠相比,两组模型小鼠GLU、CHOL、LDLC明显升高（P〈0.05）;两组模型小鼠相互比较,诱发性2型糖尿病小鼠血脂水平较高（P〈0.05）。免疫指标比较显示：除IL-2外,两组模型小鼠血清中细胞因子水平均较对照组小鼠明显升高（P〈0.05）,而db/db小鼠血清中细胞因子表达较诱发性糖尿病小鼠高,其中IL-6、IFN-γ、TNF-α具有显著性差异（P〈0.05）。结论两组2型糖尿病模型小鼠均在一定程度上模拟了人类糖尿病患者症状,但由于糖尿病产生的原因不同而存在着一定的差异,研究者可根据实际需要参照相关数据进行选择。     

糖尿病肾病动物模型的研究进展

糖尿病肾病是终末期肾衰的主要原因,也是糖尿病致命的重要原因。但是糖尿病肾病的致病机制迄今尚不完全明了,理想的动物模型无疑可对糖尿病肾病的研究提供重要线索。糖尿病肾病动物模型包括诱发性、自发性和转基因等多种类型的动物模型,各种类型的动物模型在疾病的发生发展、病理生理变化等多个方面与人类糖尿病肾病具有相似的特征。应用这些模型有助于开展对糖尿病肾病的防治、发病机理、相关药物的开发等多方面的研究。