| An ex vivo Approach to Assess Mitochondrial ROS by Flow Cytometry in AAV-tagged Astrocytes in Adult Mice
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Author:
Date:
2020-03-20
[Abstract] Mitochondrial reactive oxygen species (mROS) are naturally produced signalling molecules extremely relevant for understanding both health- and disease-associated biological processes. The study of mROS in the brain is currently underway to decipher their physiopathological roles and contributions in neurological diseases. Recent advances in this field have highlighted the importance of studying mROS signalling and redox biology at the cellular level. Neurons are especially sensitive to the harmful effects of excess mROS while astrocytic mROS have been shown to play a relevant physiological role in cerebral homeostasis and behaviour. However, given the complexity of the brain, investigating mROS formation in a specific cell-type in adult animals is methodologically challenging. Here we ...
[摘要] [摘要] 线粒体活性氧（mROS ）是天然产生的信号分子，与理解健康和疾病相关的生物过程极为相关。目前正在研究大脑中的mROS ，以了解其在神经疾病中的生理病理作用和贡献。该领域的最新进展凸显了在细胞水平研究mROS 信号传导和氧化还原生物学的重要性。神经元对过量的mROS 的有害作用特别敏感，而星形细胞的mROS对 已经显示出在脑稳态和行为中起相关的生理作用。但是，鉴于大脑的复杂性，研究成年动物中特定细胞类型中mROS的形成在方法上具有挑战性。在这里，我们提出了一种方法来具体评估星形胶质细胞中的mROS 丰度，该方法结合了i ）一种靶向策略，该策略基于在星形胶质细胞（胶质纤维酸性蛋白或GFAP）下表达绿色荧光蛋白（GFP）的腺相关病毒（AAV）载体的使用）启动子，以及ii）使用商业探针通过流式细胞术测量mROS 的强大且广泛扩展的方案。这项工作的意义在于，它允许通过易于使用的技术来选择性研究星形细胞mROS的丰度。
[背景 ] 氧化损伤与许多疾病的病因有关，包括神经退行性疾病，因为大脑是氧化应激以及与年龄相关的变化异常脆弱的组织（Cobley 等，2018; Mattson和Arumugam ...
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| Maintenance of Schmidtea mediterranea in the Laboratory
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Author:
Date:
2018-10-05
[Abstract] In the last years, planarians have emerged as a unique model animal for studying regeneration and stem cells biology. Although their remarkable regenerative abilities are known for a long time, only recently the molecular tools to understand the biology of planarian stem cells and the fundamentals of their regenerative process have been established. This boost is due to the availability of a sequenced genome and the development of new technologies, such as interference RNA and next-generation sequencing, which facilitate studies of planarian regeneration at the molecular and genetic level. For these reasons, maintain a healthy and stable planarian population in the laboratory is essential to perform reproducible experiments. Here we detail the protocol used in our laboratory to maintain ...
[摘要] 在过去的几年中，涡虫已成为研究再生和干细胞生物学的独特模型动物。 虽然它们具有显着的再生能力，但很长一段时间以来，人们已经建立了理解涡虫干细胞生物学及其再生过程基础的分子工具。 这种推动是由于测序基因组的可用性以及干扰RNA和新一代测序等新技术的发展，这些技术促进了分子和基因水平的涡虫再生研究。 由于这些原因，在实验室中保持健康稳定的涡虫种群对于进行可重复的实验至关重要。 在这里，我们详细介绍了我们实验室使用的协议，以维护最广泛作为模型的涡虫种类 Schmidtea mediterranea 。
【背景】涡虫是双侧对称的扁形动物，是超级寄主lophotroczoa的成员。有陆地，海洋和淡水涡虫。它们主要捕食受伤的昆虫，昆虫幼虫和其他无脊椎动物。涡虫是缺乏循环系统，骨骼系统和呼吸系统的三倍体和动脉粥样硬化动物（图1A）。这些动物在几天内截肢后具有恢复身体任何缺失部位的惊人能力（Reddien和Alvarado，2004; Salo，2006）;并根据环境条件和食物供应情况进行生长和驯化（Baguñá和Romero，1981）。这些特征是由于存在成体干细胞群 - 称为新生细胞 - 能够产生任何涡虫细胞类型（Reddien和Alvarado，2004; Salo，2006）。涡虫的高再生能力，具有独特的全能干细胞系统，为研究细胞更新，再生和干细胞调节提供了理想的模型。 ...
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| Investigating Neural Stem Cell and Glioma Stem Cell Self-renewal Potential Using Extreme Limiting Dilution Analysis (ELDA)
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Author:
Date:
2018-09-05
[Abstract] Glioma stem cells (GSC) grown as neurospheres exhibit similar characteristics to neural stem cells (NSC) grown as neurospheres, including the ability to self-renew and differentiate. GSCs are thought to play a role in cancer initiation and progression. Self-renewal potential of GSCs is thought to reflect many characteristics associated with malignancy, including tumor recurrence following cytotoxic therapy due to their proliferative dormancy and capacity to allow for the development of resistant tumor cell sub-clones due to mutations acquired during their differentiation. Here, we demonstrate that using extreme limiting dilution analysis (ELDA), subtle differences in the frequency of sphere-forming potential between PI3K-mutant oncogenic NSCs and non-oncogenic NSCs can be measured, in ...
[摘要] 作为神经球生长的神经胶质干细胞（GSC）表现出与作为神经球生长的神经干细胞（NSC）相似的特征，包括自我更新和分化的能力。 GSC被认为在癌症的发生和发展中起作用。 GSC的自我更新潜力被认为反映了与恶性肿瘤相关的许多特征，包括细胞毒性治疗后的肿瘤复发，这是由于它们的增殖性休眠和由于在其分化期间获得的突变而允许产生抗性肿瘤细胞亚克隆的能力。在这里，我们证明使用极限稀释分析（ELDA），可以测量PI3K-突变致癌NSCs和非致癌NSCs之间的球形成潜力频率的细微差异体外。我们进一步展示了ELDA如何在强制分化之前和之后用于细胞，以放大突变体和对照NSCs之间的球形成潜力的固有差异。最终，ELDA利用单个或少数种子干细胞自我更新，分裂和形成神经球的能力差异。重要的是，该测定还允许在不同条件下在遗传上不同的细胞之间或相同细胞之间进行比较，其中可以测试靶特异性药物或其他新型癌症干细胞疗法的影响。
【背景】胶质母细胞瘤（GBM）是最常见的脑癌之一，预后极差（Kaye和Morokoff，2014）。 ...
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