比较在体内 GP96佐剂研究中的青蛙非洲爪蟾</em
Summary
青蛙<em>非洲爪蟾</em>提供了一个有吸引力的替代非哺乳动物模型探讨热休克蛋白等作为GP96的能力，促进抗原特异性CD8 T细胞反应。我们目前的方法来研究<em>在体内</em> GP96促进皮肤和肿瘤抗原的交叉呈现。
Abstract
我们已经开发的两栖类爪蟾蟾一个独特的非哺乳动物模型来研究在一定的热休克蛋白（HSP的）的能力，如GP96，以方便陪护抗原交叉呈现，引起先天免疫和适应性的T细胞反应。爪蟾的皮 肤移植排斥反应研究的GP96引出经典MHC Ia类（Ia类）的有限制的T细胞反应的能力提供了一个极好的平台。此外， 非洲爪蟾模型系统还提供了一个有吸引力的替代小鼠探索的GP96反应生成对肿瘤有下调他们的Ia类分子，从而逃避免疫监视的能力。最近，我们开发了一种使用腹腔白细胞抗原提呈细胞（APCs）在爪蟾克隆收养细胞转移实验，并表明，GP96可以首相CD8 + T细胞在体内对未成年人的组织相容性皮肤抗原， 以及对非洲爪蟾胸腺肿瘤15反应/ 0，这并不表示Ia类分子。我们在这里描述，涉及到执行这些包括腹腔白细胞的启发，脉冲和收养的转移，以及皮肤移植和肿瘤移植实验的实验方法。此外，我们还描述的收获外周血白细胞使用流式细胞仪和增殖实验皮肤排斥和抗肿瘤反应的效应人口的进一步鉴定和分离。
Protocol
1。动物 非洲爪蟾祥煦gilli杂交LG – 6和LG – 15 isogenetic克隆是从我们的繁殖地在罗切斯特大学（http://www.urmc.rochester.edu/smd/mbi/xenopus/index.htm） 。 LG – 6和LG – 15的份额相同的杂合的MHC单倍型（A / C），但不同的次要组织相容性位点（H）。从这些克隆后代，是由雌核发育，女性所产生的二倍体卵子是由紫外线照射精子（无DNA的贡献后代）被激活。 使用…
Discussion
两栖类爪蟾是一个独特的多功能非哺乳动物模型来研究的免疫力。已经取得了许多重要的研究工具，如MHC的定义克隆LG – 6和LG – 15以及不同的细胞系和单克隆抗体及其在生物医学和免疫学研究中的广泛使用。使用这些工具，我们已经建立了在体外和体内实验研究热休克蛋白GP96如的能力，调解有力银特定抗轻微的H – Ag和抗肿瘤的T细胞反应。这个模型系统，我们可以进一步探讨在吸效应?…
Declarações
The authors have nothing to disclose.
Acknowledgements
蒂娜马丁和大卫奥尔布赖特提供的专家畜牧业衷心赞赏。这项研究得到了补助的T32 – AI – 07285（HN），美国国立卫生研究院R25 2GM064133（TCL），1R03 HD061671 – 01，R24 – AI – 059830 – 06由美国国立卫生研究院。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Reagents needed: | ||||
| Amphibian Phosphate Buffered Saline (APBS): | ||||
| NaCl, 1.15 g/L | ||||
| Na2HPO4, 0.2 g/L | ||||
| KH2PO | ||||
| 10N NaOH | ||||
| Tricaine Methane Sulfonate (TMS, MS-222) | Crescent Research Chemicals | CAS#886-86-2 | ||
| Sodium bicarbonate | Fisher Scientific | S-233-500 | ||
| Histopaque-1077 | Sigma-Aldrich | 10771 | 100ml | |
| Heparin Sodium Salt | Sigma-Aldrich | H3149-50KU | ||
| Culture medium for Xenopus 15/0 tumors [see 3 for more details]: | ||||
| Iscove DMEM basal medium | Gibco-Invitrogen | 11965 | ||
| Insulin | ||||
| Non-essential amino acids | ||||
| Penicillin-streptomycin | ||||
| Kanamycin | ||||
| Primatone | Sheffield Products Division | |||
| β2-mercaptoethanol | ||||
| NaHCO3 | ||||
| 30% double distilled water | ||||
| 5% featal bovine serum | ||||
| 20% superantant from a Xenopus kidney cell line A6 | ||||
| 0.25% of normal Xenopus serum | ||||
| Materials and Equipment: | ||||
| 50 and 15 ml conical centrifuge tubes (sterile) | ||||
| 25 gauge 5/8 Precision Glide sterile needles | BD | |||
| 18 gauge 1½ Precision Glide sterile needles | BD | |||
| 1 ml Tuberculin Slip Tip Syringe sterile | BD | |||
| 10 ml Slip Tip Syringe sterile | BD | |||
| 1.5 ml MicroCentrifuge tubes (sterile) | ||||
| 60 X 15 mm Polystyrene Petri Dishes sterile | Falcon | |||
| Razor blades | ||||
| 25 X 75 mm X 1 mm Premium Microscope Slides | Fisher Scientific | |||
| 10 cm glass petri dishes | ||||
| 9″ Pasteur Pipetes Durex Borosilicate Glass Cotton Plugged Disposable | VWR | |||
| Tygon tubing | ||||
| Two # 5 Swiss Jeweler’s Forceps | Miltex Inc. | |||
| Micro Dissecting Spring Scissors McPherson-Vannas straight cutting edge 6 mm | Roboz | |||
| Helios calipers | ||||
| Dissecting microscope | ||||
| High intensity illuminator | ||||
| Hemacytometer | ||||
| Un-bunsen burner | ||||
| 37°C shaking incubator | ||||
| Centrifuge |
Referências
- Kobel, H. R., Pasquier, D. u., L, . Hyperdiploid species hybrids for gene mapping in Xenopus. Nature. 279, 157-158 (1979).
- Robert, J., Menoret, A., Basu, S., Cohen, N., Srivastava, P. R. Phylogenetic conservation of the molecular and immunological properties of the chaperones gp96 and hsp70. Eur J Immunol. 31, 186-195 (2001).
- Robert, J., Gantress, J., Cohen, N., Maniero, G. D. Xenopus as an experimental model for studying evolution of hsp–immune system interactions. Methods. 32, 42-53 (2004).
- Chardonnens, X., Pasquier, D. u., L, . Induction of skin allograft tolerance during metamorphosis of the toad Xenopus laevis: a possible model for studying generation of self tolerance to histocompatibility antigens. Eur J Immunol. 3, 569-573 (1973).
- Du Pasquier, L., Bernard, C. C. Active suppression of the allogeneic histocompatibility reactions during the metamorphosis of the clawed toad Xenopus. Differentiation. 16, 1-7 (1980).
- Ramanayake, T., Simon, D. A., Frelinger, J. G., Lord, E. M., Robert, J. In vivo study of T-cell responses to skin alloantigens in Xenopus using a novel whole-mount immunohistology method. Transplantation. 83, 159-166 (2007).
- Robert, J., Ramanayake, T., Maniero, G. D., Morales, H., Chida, A. S., S, A. Phylogenetic conservation of glycoprotein 96 ability to interact with CD91 and facilitate antigen cross-presentation. J Immunol. 180, 3176-3182 (2008).
- Robert, J., Gantress, J., Rau, L., Bell, A., Cohen, N. Minor histocompatibility antigen-specific MHC-restricted CD8 T cell responses elicited by heat shock proteins. J Immunol. 168, 1697-1703 (2002).
- Morales, H., Robert, J. In vivo and in vitro techniques for comparative study of antiviral T-cell responses in the amphibian Xenopus. Biol Proced Online. 10, 1-8 (2008).
- Maniero, G. D., Robert, J. Phylogenetic conservation of gp96-mediated antigen-specific cellular immunity: new evidence from adoptive cell transfer in xenopus. Transplantation. 78, 1415-1421 (2004).
Tags
Gp96 AdjuvanticityComparative In Vivo StudyFrog Xenopus LaevisHeat Shock ProteinsChaperoned AntigensInnate And Adaptive T Cell ResponsesXenopus Skin Graft RejectionMHC Class Ia Restricted T Cell ResponsesTumors Down-regulating Class Ia MoleculesAdoptive Cell Transfer AssayPeritoneal LeukocytesAntigen Presenting Cells (APCs)CD8 T Cell ResponsesMinor Histocompatibility Skin AntigensXenopus Thymic Tumor 15/0MethodologyElicitationPulsingAdoptive TransferSkin Graft Transplantation AssayTumor Transplantation AssayHarvesting Peripheral Blood LeukocytesFlow CytometryProliferation Assays
Citar este artigo
Nedelkovska, H., Cruz-Luna, T., McPherson, P., Robert, J. Comparative in vivo Study of gp96 Adjuvanticity in the Frog Xenopus laevis. J. Vis. Exp. (43), e2026, doi:10.3791/2026 (2010).