| Buoyant Density Fractionation of Small Extracellular Vesicle Sub-populations Derived from Mammalian Cells
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Author:
Date:
2020-08-05
[Abstract] Small extracellular vesicles (sEVs) encompass a variety of distinct vesicles that are secreted to the extracellular space. Many methodologies currently used for EV isolation (e.g., differential ultracentrifugation concluding in a high-speed pellet, precipitation by macromolecular crowding agents or size excusion chromatography–SEC) do not fractionate distinct sEV sub-populations. Samples obtained by the aforementioned methods are usually used for characterization and physiological studies. However the fraction that contains the molecule of interest or is the carrier of a specific activity is unknown. Therefore isolating distinct sEV sub-populations is critical to understand EV function. The goal of this procedure is to purify distinct sEV sub-populations based on slight ...
[摘要] [摘要] 小细胞外小泡（sEVs）包括分泌到细胞外空间的各种不同的小泡。目前用于EV分离的许多方法（例如，高速颗粒中的差速超速离心、大分子拥挤剂沉淀或尺寸排除色谱法）没有分离不同的sEV亚群。通过上述方法获得的样品通常用于表征和生理学研究。然而，包含感兴趣分子或特定活性载体的部分是未知的。因此，分离不同的sEV亚群对于理解EV功能至关重要。该程序的目的是基于它们浮力密度的微小差异来纯化不同的sEV亚群。此外，该技术还允许从高速颗粒中共同分离的无囊泡RNA蛋白复合物中或通过使用拥挤剂来纯化sEVs。该方案描述了用于收集sEV的哺乳动物细胞的培养、sEV沉淀、sEV亚群的浮力密度分馏和sEV标记的免疫印迹。该方法可用于分离由多种哺乳动物细胞产生的不同的sEV亚群。
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| Modeling NOTCH1 driven T-cell Acute Lymphoblastic Leukemia in Mice
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Author:
Date:
2020-05-20
[Abstract] T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that arises from transformation of T-cell primed hematopoietic progenitors. Although T-ALL is a heterogenous and molecularly complex disease, more than 65% of T-ALL patients carry activating mutations in the NOTCH1 gene. The majority of T-ALL–associated NOTCH1 mutations either disrupt the negative regulatory region, allowing signal activation in the absence of ligand binding, or result in truncation of the C-terminal PEST domain involved in the termination of NOTCH1 signaling by proteasomal degradation. To date, retroviral transduction models have relied heavily on the overexpression of aggressively truncated variants of NOTCH1 (such as ICN1 or ΔE-NOTCH1), which result in ...
[摘要] [摘要 ] T细胞急性淋巴细胞白血病（T-ALL）是一种侵袭性血液恶性肿瘤，其起源于T细胞引发的造血祖细胞的转化。尽管T-ALL是一种异质且分子复杂的疾病，但超过65％的T-ALL患者在NOTCH1 基因中带有激活突变。大多数与T-ALL相关的NOTCH1 突变要么破坏负调控区，允许在没有配体结合的情况下激活信号，要么导致蛋白酶体降解终止NOTCH1信号终止所涉及的C末端PEST域被截短。迄今为止，逆转录病毒转导模型在很大程度上依赖于侵袭性截短的变种的过度表达。 NOTCH1 （例如ICN1或ΔE-NOTCH1）可导致信号传导的超生理水平，并且在人类T-ALL中很少见。当前方案描述了小鼠骨髓分离，造血干细胞和祖细胞（HSC）富集，然后逆转录病毒转导的致癌突变体形式的NOTCH1受体（NOTCH1-L1601P-ΔP）的方法，该方法与获功能突变最常见于患者样品中。组成型活性NOTCH1的这种强制表达的标志是胸腺外未成熟T细胞发育的瞬时波，此波在致癌性转化为T-ALL之前。此外，该方法通过允许白血病细胞与微环境之间的串扰来模拟体内白血病的转化和进展，这是基于细胞系的体外研究无法解释的一个方面。因此，HSC转导和移植模型更真实地概括了人类疾病的发展，为进一步的体内和离体功能研究提供了高度全面和通用的工具。
[背景 ] ...
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