Adhesion of Enteroaggregative E. coli Strains to HEK293 Cells
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Author:
Date:
2018-04-20
[Abstract] Enteroaggregative Escherichia coli (EAEC) is a recognized cause of acute diarrhea among both children and adults worldwide. EAEC strains are characterized by the presence of aggregative adherence fimbriae (AAF), which play a key role in pathogenesis by mediating attachment to the intestinal mucosa and by triggering host inflammatory responses. The aggregative adherence fimbria II (AAF/II) is the most important adherence factor of EAEC prototype strain 042 (EAEC042) to intestinal cells. Multiple receptors for AAF/II on epithelial cells have been identified including the transmembrane signaling mucin Muc1. This protocol describes a method to measure adherence of EAEC strains to HEK293 cells expressing the Muc1 glycoprotein.
[摘要] 肠道集聚性大肠杆菌(EAEC)是全球儿童和成人急性腹泻的公认原因。 EAEC菌株的特征在于存在聚集粘附菌毛(AAF),其通过介导与肠粘膜的附着和通过引发宿主炎症反应而在发病机制中起关键作用。 聚合粘附菌毛II(AAF / II)是EAEC原型菌株042(EAEC042)对肠细胞最重要的粘附因子。 已经鉴定了上皮细胞上AAF / II的多种受体,包括跨膜信号传导粘蛋白Muc1。 该协议描述了测量EAEC菌株对表达Muc1糖蛋白的HEK293细胞的依从性的方法。
【背景】EAEC是世界范围内地方性和流行性腹泻病的重要原因。尽管发展中国家儿童腹泻最常见,但EAEC还与免疫受损成人腹泻,旅行者和工业化国家的食源性疾病有关,例如由志贺毒素(Stx)2a型产生的大致致命爆发2011年在北欧的血清型O104:H4的EAEC菌株(Harrington等人,2006; Rasko等人,2011)。 EAEC发病机制由生物体粘附肠细胞,产生肠毒素和细胞毒素并最终诱导炎症的能力决定(Harrington等,2006)。 EAEC对肠细胞的依从性由AAF菌毛粘附素介导(Czeczulin等人,1997)。迄今为止,已经描述了至少5种AAF菌毛的变体,全部编码在范围为55至65MDa的毒力质粒中(Jonsson等人,2015)。 ...
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A 3D Culture System of Human Immortalized Myometrial Cells
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Author:
Date:
2016-10-20
[Abstract] Myometrium forms the middle layer of the uterus and is mainly composed of the smooth muscle cells. The cells in vitro are usually grown in a single layer (2-dimensional; 2D) format, whereas in vivo cells are structured in an extracellular matrix scaffolding that allows the cells to communicate and respond to environmental cues. We have developed human myometrium and leiomyoma 3-dimensional (3D) culture, wherein the cells retain their molecular characteristics and respond to environmental cues (Malik and Catherino, 2012; Malik et al., 2014).
[摘要] 子宫肌层形成子宫的中间层,主要由平滑肌细胞组成。细胞在体外通常生长在单层(2维; 2D)格式中,而体内细胞在细胞外基质支架中结构化,其允许细胞沟通和响应环境线索。我们已经开发了人子宫肌瘤和平滑肌瘤3维(3D)培养物,其中细胞保留其分子特征并响应环境线索(Malik和Catherino,2012; Malik等人,2014)。 br /> [背景] 在过去十年中,随着更多实验室从使用人工2D格式的细胞培养转移到3D细胞培养模型系统,观察到一定的转变,其中细胞生长在允许它们附着并获得更生理结构的基质中。该模型系统为细胞提供更自然的分化状态,并且培养的细胞在体内形成组织样环境。这是一个详细的协议为myometrium 3D细胞培养生长胶原-1矩阵,修改从Malik和Catherino(2012年)。
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Assessment of Mitochondrial DNA Content and Mass in Macrophages
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Author:
Date:
2016-05-05
[Abstract] Mitochondria are essential regulators in not only ATP generation and metabolic reprogramming but also the generation of reactive oxygen species (ROS) in response to pathogenic stimuli. During exposure to environmental stresses including oxidative stress, exercise, cell division and caloric restriction, mitochondria can be divided to increase mitochondrial number, size, and mass. Moreover, mitochondrial biogenesis has a crucial role in the resolution of inflammation through preserving metabolic function. Recently, diverse biochemical methods have been utilized to evaluate activity of mitochondrial biogenesis. In this protocol, we will describe an in vitro assay to measure mitochondrial DNA content and mass. Quantitative real-time PCR analysis for determination of mitochondrial DNA content ...
[摘要] 线粒体是不仅ATP生成和代谢重新编程,而且响应病原性刺激的活性氧(ROS)的生成的必要调节器。在暴露环境应激,包括氧化应激,运动,细胞分裂和热量限制期间,线粒体可以分为增加线粒体数量,大小和质量。此外,线粒体生物发生在通过保护代谢功能解决炎症中具有关键作用。最近,不同的生物化学方法已被用来评估线粒体生物发生的活性。在这个协议,我们将描述一个体外测定来测量线粒体DNA含量和质量。用于测定线粒体DNA含量的定量实时PCR分析是添加流式细胞术或共聚焦显微镜用于评价线粒体质量的有力工具。在一起,这些协议可以提供线粒体研究的重要信息。
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