本视频展示了胎儿胸腺的解剖与摘除，以及经2-dGuo处理的胸腺体外培养的制备过程。
本文内容
勘误通知
Important: There has been an erratum issued for this article. View Erratum Notice
摘要
摘要
在胸腺中，发育中的T细胞前体与包括皮质和髓质上皮细胞在内的基质细胞之间的相互作用，已被证实对形成功能健全的T细胞库具有关键作用。然而，胸腺内T细胞发育过程的复杂性 体内 可能会限制对单个细胞组分和特定发育阶段的分析。 体外 培养系统为研究多种复杂的细胞过程提供了便捷的途径。胸腺器官培养系统是一种广泛应用的方法，可用于在明确条件下研究T细胞的胸腺内发育过程 体外在此，我们描述了一种系统，该系统通过在2-脱氧鸟苷（2-deoxyguanosine）中预先进行器官培养，可去除小鼠胚胎胸腺小叶中的内源性造血成分，该化合物对造血细胞具有选择性毒性。该技术不仅为研究胸腺微环境在T细胞发育与选择中的作用提供了易于获取的胸腺基质细胞来源，也为进一步的实验方法奠定了基础，包括无淋巴细胞胸腺小叶的重建 体外 含有明确的造血成分，将无淋巴细胞的胸腺移植到受体小鼠体内，以及重建胸腺类器官培养物的形成。（本文基于首次发表于《Methods in Molecular Biology》2007年第380卷第185–196页的研究工作。）
方案
有关胸腺器官离体培养制备的更多信息，请访问 Springer Protocols。
披露
作者无任何利益冲突需要披露。
重印与许可
勘误
Formal Correction: Erratum: Preparation of 2-dGuo-Treated Thymus Organ Cultures
Posted by JoVE Editors on 4/01/2012. Citeable Link.
A correction was made to: Preparation of 2-dGuo-Treated Thymus Organ Cultures. A revised abstract was republished due to a publisher error. The abstract was corrected to:
In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play a key role in the development of a functionally competent T-cell pool. However, the complexity of T-cell development in the thymus in vivo can limit analysis of individual cellular components and particular stages of development. In vitro culture systems provide a readily accessible means to study multiple complex cellular processes. Thymus organ culture systems represent a widely used approach to study intrathymic development of T-cells under defined conditions in vitro. Here we describe a system in which mouse embryonic thymus lobes can be depleted of endogenous haemopoeitic elements by prior organ culture in 2-deoxyguanosine, a compound that is selectively toxic to haemopoeitic cells. As well as providing a readily accessible source of thymic stromal cells to investigate the role of thymic microenvironments in the development and selection of T-cells, this technique also underpins further experimental approaches that include the reconstitution of alymphoid thymus lobes in vitro with defined haemopoietic elements, the transplantation of alymphoid thymuses into recipient mice, and the formation of reaggregate thymus organ cultures. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).
from
In the thymus, immature CD4+8+ thymocytes expressing randomly rearranged T-cell receptor α- and b-chain genes undergo positive and negative selection events based on their ability to recognize self-peptide/major histocompatibility complex (MHC) molecules expressed by thymic stromal cells. In vivo analysis of the role of thymic stromal cells during intrathymic selection is made difficult by the cellular complexity of the thymic microenvironment in the steady-state adult thymus, and by the lack of appropriate targeting strategies to manipulate gene expression in particular thymic stromal compartments. We have shown that the thymic microenvironment can be readily manipulated in vitro through the use of reaggregate thymus organ cultures, which allow the preparation of three-dimensional thymus lobes from defined stromal and lymphoid cells. Although other in vitro systems support some aspects of T-cell development, reaggregate thymus organ culture remains the only in vitro system able to support efficient MHC class I and II-mediated thymocyte selection events, and so can be used as an effective tool to study the cellular and molecular regulation of positive and negative selection in the thymus.
