| Primary Cultures from Human GH-secreting or Clinically Non-functioning Pituitary Adenomas
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Author:
Date:
2018-04-05
[Abstract] Pituitary adenomas are among the more frequent intracranial tumors usually treated with both surgical and pharmacological–based on somatostatin and dopamine agonists–approaches. Although mostly benign tumors, the occurrence of invasive behaviors is often detected resulting in poorer prognosis. The use of primary cultures from human pituitary adenomas represented a significant advancement in the knowledge of the mechanisms of their development and in the definition of the determinants of their pharmacological sensitivity. Moreover, recent studies identified also in pituitary adenomas putative tumor stem cells representing, according to the current hypothesis, the real cellular targets to eradicate most malignancies. In this protocol, we describe the procedure to establish primary cultures ...
[摘要] 垂体腺瘤是更常见的颅内肿瘤之一，通常用基于生长抑素和多巴胺激动剂手术的手术和药物治疗。 虽然多为良性肿瘤，但侵入性行为的发生常常被检测到，导致预后较差。 来自人类垂体腺瘤的原代培养物的使用代表了对其发育机制的知识以及其药理敏感性决定因素的定义方面的显着进步。 此外，最近的研究也在垂体腺瘤中发现了假定的肿瘤干细胞，根据目前的假设，它代表了根除大多数恶性肿瘤的真实细胞靶标。 在这个协议中，我们描述了从人垂体腺瘤建立原代培养的程序，以及如何选择，体外扩增和表型鉴定推定的垂体腺瘤干细胞。
【背景】垂体腺瘤是最常见的颅内肿瘤之一（高达15％），横断面研究发现每100,000名居民中约有90例发病，其中绝大多数为30岁以上的成年人。大约10％的未经选择的垂体在尸检时进行了检查（即考虑到之前未诊断为垂体疾病的受试者的垂体）（ ，Molitch，2017）。尽管通常为良性肿瘤，但垂体腺瘤的处理可因与激素分泌过多相关的临床综合征或发展以治疗抗性，高增殖率，快速复发和绒毛外侵袭为特征的侵袭行为而复杂化（Carreno等人，2017）。成年垂体干细胞的持续存在（Florio，2011）导致垂体腺瘤（以及可能的其他良性瘤形成）的发展可以源自具有干细胞特性（主要是自我更新和分化）的肿瘤细胞的亚群能力），正如已经建立的恶性固体和血液肿瘤一样。
最近的实验证据表明，癌症干细胞（CSC）范例也适用于人和小鼠垂体腺瘤（Donangelo等人，2014; ...
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| Killer Cell Ig-like Receptors (KIR)-Binding Assay for Tumor Cells
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Author:
Date:
2016-09-20
[Abstract] Natural killer (NK) cells play key roles in innate and adaptive immune responses against virus and tumor cells. Their function relies on the dynamic balance between activating and inhibiting signals through receptors that bind ligands expressed on target cells. The absence of inhibitory receptor engagement with their ligands and the presence of activating signals transmitted by activating receptors interacting with specific ligands, leads to NK cell activation (Lanier, 2005; Raulet et al., 2001). Thus, the balance of the ligands expressed for inhibitory and activating receptors determines whether NK cells will become activated to kill the target cells. This protocol allows to assign a precise ligand specificity to any given receptor on NK cells. Thus, if a tumor cell expresses ...
[摘要] 自然杀伤（NK）细胞在针对病毒和肿瘤细胞的先天和适应性免疫应答中发挥关键作用。它们的功能依赖于通过结合靶细胞上表达的配体的受体在激活和抑制信号之间的动态平衡。不存在与其配体的抑制性受体接合和通过活化受体与特异性配体相互作用传递的激活信号的存在导致NK细胞活化（Lanier，2005; Raulet等人，2001）。因此，针对抑制性和激活受体表达的配体的平衡决定了NK细胞是否将被激活以杀死靶细胞。该协议允许给NK细胞上的任何给定受体分配精确的配体特异性。因此，如果肿瘤细胞表达配体，则该方案将允许评价其与特异性受体的相互作用。特别地，通过与HLA I类重链残基和结合肽的氨基酸残基的直接接触，杀伤细胞免疫球蛋白（Ig）样受体（KIR）识别它们的配体（HLA I类分子）。该方案将允许测试氨基酸取代或其他突变对KIR与HLA I型结合的影响。我们使用该方案描述ERAP1（MHC I类抗原加工的关键组分）在调节NK细胞中的作用通过控制抑制性受体的接合来控制细胞功能（Cifaldi等人，2015）。
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| Peptide Loading on MHC Class I Molecules of Tumor Cells
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Author:
Date:
2016-09-20
[Abstract] MHC class I molecules present peptides to cytotoxic T cells allowing the immune system to scan for intracellular pathogens and mutated proteins. The generation of antigenic peptides is a multistep process that ends in the endoplasmic reticulum (ER). Only peptides with the right length and sequence will bind nascent MHC class I molecules in the ER. This protocol allows for detachment of the endogenous peptides bound to MHC class I molecules by preserving them for the binding of high affinity synthetic peptides. The complete dissociation of endogenous peptides by mild acid treatment as well as the binding of synthetic peptides to MHC class I molecules will be evaluated measuring HLA class I molecules express on the cell surface by flow cytometry. The mouse antibody W6/32 which recognizes ...
[摘要] MHC I类分子将肽呈递到细胞毒性T细胞，允许免疫系统扫描胞内病原体和突变蛋白。抗原肽的产生是以内质网（ER）结束的多步过程。只有具有正确长度和序列的肽将结合ER中的新生MHC I类分子。该方案允许通过保持结合MHC I类分子的内源肽与高亲和力合成肽的结合而脱离。通过温和酸处理以及合成肽与MHC I类分子的结合来评价内源肽的完全解离，通过流式细胞术测量在细胞表面上表达的HLA I类分子。识别β2m相关HLA-A，-B，-C，-E和-G重链的小鼠抗体W6/32适合于此提议。表达表面HLA I类分子的任何肿瘤细胞系适于测定。另一个重要方面是知道肿瘤细胞系的HLA I类分型以允许选择已知的高亲和力肽。
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