Bereiding van 2-dGuo behandelde Thymus Organ culturen

Biology
 

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Summary

Deze video toont de dissectie en verwijdering van de foetale thymus en de voorbereiding van de ex vivo kweken van 2-dGuo behandeld thymus.

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Jenkinson, W., Jenkinson, E., Anderson, G. Preparation of 2-dGuo-Treated Thymus Organ Cultures. J. Vis. Exp. (18), e906, doi:10.3791/906 (2008).

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Abstract

In de thymus, onvolwassen CD4 +8 + thymocyten uiten willekeurig herschikte T-cel-receptor α-en b-keten genen ondergaan positieve en negatieve selectie gebeurtenissen op basis van hun vermogen om self-peptide/major histocompatibility complex (MHC) moleculen uitgedrukt door de thymus te herkennen stromale cellen. In vivo analyse van de rol van de thymus stromale cellen tijdens intrathymic selectie wordt bemoeilijkt door de cellulaire complexiteit van de thymus micro-omgeving in de steady-state voor volwassenen thymus, en door het ontbreken van passende richten op strategieën om de genexpressie in het bijzonder thymus stromale compartimenten te manipuleren. We hebben aangetoond dat de thymus micro-omgeving kan gemakkelijk worden gemanipuleerd in vitro door het gebruik van reaggregate thymus orgaan culturen, die de voorbereiding van de drie-dimensionale thymus lobben van toegezegd stromale en lymfoïde cellen mogelijk te maken. Hoewel de andere in-vitro-systemen ondersteunen een aantal aspecten van de T-cel ontwikkeling, reaggregate thymus orgelcultuur blijft de enige in vitro systeem in staat om een ​​efficiënte ondersteuning van MHC klasse I en II-gemedieerde thymocyt selectie van evenementen, en kunnen dus gebruikt worden als een effectief instrument om te studeren de cellulaire en moleculaire regulatie van positieve en negatieve selectie in de thymus.

Protocol

Ga naar Springer Protocols voor meer informatie over de voorbereiding van de ex vivo thymus orgaan culturen.

Erratum

Formal Correction: Erratum: Preparation of 2-dGuo-Treated Thymus Organ Cultures
Posted by JoVE Editors on 04/01/2012. 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.

Comments

2 Comments

  1. This is interesting detail technique for dissecting, culturing thymus embryo to study the effect of ² d-Guo. The authors may be interesting to know about the effect of dGuo on islets. Please see those two abstracts of published articles.     1: Transplantation. 1991 May; 51(5):967-71. Links   Prolongation of allograft survival in diabetic rats treated with cyclosporine by deoxyguanosine pretreatment of pancreatic islets of Langerhans. al-Abdullah IH, Kumar AM, al-Adnani MS, Abouna GM. Department of Organ Transplantation and Pathology, Faculty of Medicine, Kuwait University, Safat. In vitro pretreatment of islets of Langerhans with deoxyguanosine (dGuo) has been shown to be effective for the prolongation of islet allograft survival in rats. [This study evaluates the effect of pretreatment of islets with dGuo transplanted into CsA-treated recipients.] Transplantation of dGuo-treated islets from Wistar rats into diabetic hooded (PVG) rats resulted in 36% graft survival without immunosuppression (dGuo-group) and 89% islet survival after a short course of cyclosporine was used in recipients (dGuo + CsA group). In contrast, transplantation of untreated islets into rats without immunosuppression (controls) and with CsA (CsA group) immunosuppression resulted in 0 and 56% survival, respectively. The differences in graft survival between dGuo versus control group (P less than 0.001), (dGuo + CsA) versus control group (P less than 0.0001), and CsA versus control group (P less than 0.00²) are statistically significant. Donor-strain skin-graft challenge failed to induce rejection of transplanted normoglycemic rats in (dGuo) and (dGuo + CsA) groups. The results indicate that a state of immunologic unresponsiveness may have been induced in the recipients of dGuo-treated islets, and further treatment with CsA synergistically prolongs islet survival in fully mismatched rats. PMID: ²031²80 [PubMed - indexed for MEDLINE]           1: Diabetes Res. 1991 Aug;17(4):181-7. Links   Improvement in allograft survival of islets of Langerhans by pretreatment with deoxyguanosine. al-Abdullah IH, Kumar MS, al-Adnani MS, Abouna GM. Department of Surgery, Hahnemann University, Philadelphia, Pennsylvania 1910². In vitro pretreatment of isolated islets of Langerhans prior to transplantation with deoxyguanosine (dGuo) was found to be effective in improving the survival in fully allogeneic diabetic rats (Wistar----PVG). Post transplant immunosuppression was not used. Islets pretreated with various concentrations of dGuo, 1, 1.35, 1.5 and ² microM dGuo per islet showed a graft survival of 9, 36, 9 and 14% respectively. PMID: 18²3639 [PubMed - indexed for MEDLINE]  

    Reply
    Posted by: Anonymous
    February 9, 2009 - 12:27 PM
  2. hello: it is wonderfull .and i is just doing some research using this method ! thank you very much!
    but i just have some techincal problem to ask you ,that is how to prepare the ²d-Guo stock solution ? because i am the first year in immunology research and i do not how to disvŒ the ²d-Guo in PBS ! is there and key prosedure to do it ?when i put the ²d-Guo to the PBS ,it dŒs not dissove at all ! i post this question for you , may you can reply me ,thank you very much ! my email is : xueliangxiu@hotmail.com

    Reply
    Posted by: xueliang x.
    June 3, 2009 - 9:26 AM

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