| Generation of Mouse Primary Hypothalamic Neuronal Cultures for Circadian Bioluminescence Assays
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Author:
Date:
2021-03-05
[Abstract] An endogenous circadian clock system enables organisms to adapt to time-of-day dependent environmental changes. In consequence, most physiological processes exhibit daily rhythms of, e.g., energy metabolism, immune function, sleep, or hormone production. Hypothalamic circadian clocks have been identified to play a particular role in coordinating many of these processes. Primary neuronal cultures are widely used as a physiologically relevant model to study molecular events within neurons. However, as circadian rhythms include dynamic molecular changes over longer timescales that vary between individual cells, longitudinal measurement methods are essential to investigate the regulation of circadian clocks of hypothalamic neurons. Here we provide a protocol for generating primary ...
[摘要] [摘要]内源性生物钟系统使生物能够适应与时间相关的环境变化。结果，大多数生理过程表现出例如能量代谢，免疫功能，睡眠或激素产生的每日节律。下丘脑生物钟已被确认在协调许多这些过程中起特定作用。 原代神经元文化被广泛用作研究神经元内分子事件的生理相关模型。然而，由于昼夜节律包括较长时间范围内的动态分子变化，而这种变化在各个细胞之间会有所不同，因此纵向测量方法对于研究下丘脑神经元昼夜节律的调节至关重要。在这里，我们提供了用于生成表达昼夜节律性荧光素酶报道基因的下丘脑神经元文化的协议。通过执行生物发光测量，此类报告细胞可用于以高时间分辨率纵向监测细胞昼夜节律。
[背景]为了适应重复在其环境中的时间-日期依赖性变化，许多生物已开发出一种内源性生物钟系统调节行为和生理过程的24小时的节律（夏尔马，2003）。在哺乳动物中，一个昼夜节律性起搏器主要位于下丘脑上视交叉上核（SCN）。它与外部时间协调整个身体的细胞时钟调节。睡眠，食欲和新陈代谢的每日模式由下丘脑神经元中的细胞昼夜节律调节（Cedernaes等，2019）。
在哺乳动物细胞中，昼夜节律时钟由互锁的转录-翻译反馈环（TTFL）组成。在核心TTFL中，转录因子昼夜运动输出周期kaput（CLOCK）和脑和肌肉芳基碳氢化合物受体核转运蛋白样蛋白1（BMAL1或ARNTL）激活其自身阻遏物，周期（PER1-3）和隐色蛋白的表达（CRY1 ...
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| Evolution of Escherichia coli to Macrophage Cell Line
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Author:
Date:
2014-09-05
[Abstract] The genomes of species of Escherichia coli (E. coli) show an extraordinary amount of diversity, which include commensal strains and strains belonging to different pathovars. Many strains of E. coli, which can cause mild or severe pathologies in humans, have a commensal ancestor. Understanding the evolutionary changes that can lead to a transition from commensal to pathogen is an important task, which requires integration of different methodologies. One method is experimental evolution of bacteria, in controlled environments, that mimic some of the selective pressures, likely to be important during the transition to pathogenesis. The success of such a transition will depend, at least partially, on ability of E. coli to adapt to the presence of cells of ...
[摘要] 大肠杆菌物种的基因组（大肠杆菌）显示出非凡的多样性，其包括共生菌株和属于不同病原体的菌株。许多菌株。大肠杆菌，其可引起人类的轻度或严重病理，具有共生祖先。了解可导致从共生转变为病原体的进化变化是一项重要任务，需要整合不同的方法。一种方法是在受控环境中的细菌的实验进化，其模拟一些选择性压力，在向发病过渡期间可能是重要的。这种转变的成功将至少部分地取决于E的能力。大肠杆菌以适应免疫系统细胞的存在。在这里，我们描述了用于进行共生应变的实验进化的方案。大肠杆菌，在充分研究的K12的衍生物，在先天免疫系统的细胞施加的恒定选择压力下，特别是RAW 264.7鼠巨噬细胞细胞系。
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| Isolation and 3-dimensional Culture of Primary Murine Intestinal Epithelial Cells
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Author:
Date:
2014-05-20
[Abstract] The intestine, together with skin and blood, belongs to the organs with the highest cell turnover, which makes it a perfect model to study cellular processes, such as proliferation and differentiation. Epithelial cell turnover in intestine is possible due to the presence of intestinal stem cells, which are located at the bottom of the crypt. Here, we recapitulate a detailed protocol for the isolation and culture procedures of primary epithelial intestinal cells in a three - dimensional (3D) in vitro system, described for the first time by Hans Clevers group (Sato et al., 2009). This specific 3D culture preserves intestinal stem cells, which give rise to differentiated progeny for example goblet cells. The culture has many applications and represents a useful model to ...
[摘要] 肠道与皮肤和血液一起属于具有最高细胞更新的器官，这使得它成为研究细胞过程如增殖和分化的完美模型。由于存在位于隐窝底部的肠干细胞，肠上皮细胞更新是可能的。在这里，我们重述了在三维（3D）体外系统中原发性上皮肠细胞的分离和培养程序的详细方案，首次由Hans Clevers组（Sato > et al。，2009）。这种特定的3D培养物保留肠干细胞，其产生分化的子代，例如杯状细胞。该培养物具有许多应用并且代表研究干细胞生物学，上皮细胞再生和移植研究的有用模型。此外，呈现的3D文化可用于调查肠上皮细胞的屏障功能，以及上皮细胞和基质细胞之间的异型细胞相互作用。
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