中国科学院昆明动物研究所机构知识库

本论文包括两部分相互独立的内容。第一部分主要内容是使用光纤进行活体 荧光记录技术的开发和应用。随着基因技术的发展，基因转染技术已广泛应用于 动物实验和临床研究。病毒作为外源基因的一种载体，其表效率在基因转染中发 挥着关键作用。通常对于病毒载体的表达效率的检测不得不通过处死动物以获取 组织切片，然后检测报告基因如荧光蛋白的表达情况来进行，但这对于实验周期 长，过程复杂且非常珍贵的猕猴等非人灵长类（NHP）动物来说非常难以实行。 实验人员更希望在实验进程中了解基因的表达情况，这样有利于控制实验进程并 提高实验效率。此外，目前应用的病毒载体种类，亚形及变体繁多，很多类型的 病毒在 NHP 中枢神经系统的表达效率和时程是未知数。为了帮助解决这些问题， 作者开发了一套完整的活体荧光记录装置并建立了相关的使用方法，用于检测猕 猴病毒转基因实验中病毒是否表达良好以及探究一些病毒载体在猕猴脑组中的 表达特征，如表达时程等。该装置以光纤作为介质进行荧光激发和收集，主要包 括一台 LED 光源或激光器，滤光镜和用于记录信号的光谱仪，该装置可用于动 物脑组织不同深度的荧光记录。作者验证了该装置及方法的可靠性，并在体外， 活体大鼠脑和猕猴脑组织中均记录到荧光信号，此外还绘制了几种病毒载体在猕 猴脑组织中的表达曲线。第二部分是关于以甲醛诱导猕猴产生阿尔茨海默症 （AD），并在此基础上探究百里醌（Tq）和白藜芦醇（Res）能否对甲醛的诱导 产生的 AD 病理特征进行逆转。甲醛是一种广泛存在于自然界及人们生活中的小 分子物质。尽管正常人体内具有甲醛的代谢机制，但过量的甲醛对人体具有神经 毒性。相关研究表明体内甲醛的含量与 AD 有紧密相关性。AD 病人体内的甲醛 含量高于正常人，在细胞和动物实验中也表明甲醛能诱发 Aβ 聚集及 Tau 蛋白的 磷酸化。Tq 和 Res 是自然界两种具有广泛抗氧化，抗免疫等保护作用的药物。 由于这些分子发挥作用时涉及到体内众多生理过程，其中包含神经保护的机制， 作者猜测 Tq 和 Res 可能具有抵抗 AD 病程的作用。为了证实这一假设，作者以 甲醛诱导猕猴 AD 模型，并给猕猴口服 Tq 和 Res，然后在给药过程中，通过观 察 AD 相关的经典病理特征的变化来判断 Tq 和 Res 的逆转作用。

The thesis includes two parts independently. The first part mainly involves the development and application of utilizing optical fiber to record fluorescence in vivo. With the development of gene technologies, gene transfection has been wildly used in animal experiments and clinic research. Virus is a kind of vector for exogenous genes, which the expression effect plays a critical function in gene transfection. Usually, it has to kill animals and getstissue slice to check the report genessuch as fluorescent proteins so that can examine the expression of virus. But it is definitely inconvenient for precious Non-Human primate (NHP) such as rhesus which combines long-term experiment and complicate procedure. Experimenters more like to find out the viral expressive efficiency during experimental process, which benefits to control the experimental stage and improve the experimental efficiency. Besides, viral vectors that applied currently has many species, subtypes and variants, the expressive efficiency process of many kinds of viral vectors is not clear in the central nerve system of NHP. To help to solve these problems, we developed a fluorescent record device and method in vivo to detect the expression of viral vector in gene transfection of rhesus and to research the expressive feature of some viral vectors, such as expressive circle. The device and method uses optical fiber as a media to fluorescence exciting and collecting, which mainly include a LED illuminant or laser equipment, optical filter and a spectrometer, with the application to fluorescence record in different depth. We confirmed the validation of the device and method, and we not only recorded fluorescent signal in brain of rats and rhesus successfully but also drew expressive curves along with different expressive periods of several viral vectors. The second part is about utilize formaldehyde to induce the Alzheimer’s disease (AD) in rhesus, based on which to explore whether Thymoquinone (Tq) and Resveratrol (Res) could reverse the effect of formaldehyde. Formaldehyde is a wildly exist small molecule in nature. Though normal people have metabolic mechanism of Formaldehyde in body, excessive formaldehyde Abstract III still have many physiologic toxicity, include neural toxicity. Relative researches have demonstrate that the content of formaldehyde is closely relate to AD. Not only the Formaldehyde is higher in AD patients than normal people, but also formaldehyde could induce Aβ aggregation and Tau phosphorylation in cell assay as well as animal assy. Thymoquinone (Tq) and Resveratrol (Res) are two kinds of nature medicine, with a comprehensive protect effect involve anti-oxidation and anti-immune, etc. Because the function of the two molecules involve many physiological procession that may include neural protect mechanism, we suppose that Tq and Res have the function to resist the AD procession. To confirm this hypothesis, we utilize formaldehyde to induce AD in rhesus monkeys and give Tq and Res to monkeys by oral administration. Then we observe the change of classical pathological feature of AD to check the reverse effect of Tq and Res during administration.