The role of pro-apoptotic BH3-only proteins in survival and differentiation of lymphocytes

促凋亡 BH3-only 蛋白在淋巴细胞存活和分化中的作用

• 批准号: 288787880
• 负责人: Professor Dr. Georg Häcker
• 金额: --
• 依托单位: Department für Medizinische Mikrobiologie, Virologie und Hygiene
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/288787880?language=en
• 关键词: role; pro; apoptotic; BH3; only

Mitochondrial apoptosis is a key regulatory mechanism during differentiation and life of haematopoietic cells. Mitochondrial apoptosis is governed by the Bcl-2-family of proteins; within this family the group of eight BH3-only proteins are initiators of apoptosis, which by their expression levels and possibly their activation regulate apoptosis. In immune cells, Bim is the most prominent and important BH3-only protein but a number of other BH3-only proteins (especially Puma, Bmf and Noxa) appear to play an incompletely understood role. We have learned much about mitochondrial apoptosis in immune cells especially from gene-deficient mouse models. However, further progress is slow in particular because of the limitations in obtaining the necessary cellular material. We have established a cell model where mouse haematopoietic progenitor cells can be expanded in vitro and differentiated into myeloid cells as well as into lymphocytes in vitro and in vivo. We have established such progenitor cells from mice double deficient in BH3-only proteins (Bim/Puma, Bim/Bmf, Bim/Noxa) and have found intriguing apoptosis defects in some situations. We here propose to use these cells, and to establish additional cell lines carrying triple-deficiencies for BH3-only proteins, in order to analyse the contribution of BH3-only proteins to development, maintenance and apoptosis sensitivity especially in B and T lymphocytes. In vitro, progenitors as well as "B cells" and "T cells" of the various genotypes will be tested for their apoptotic response (B cells can from the progenitors be differentiated in vitro to IgM-positive cells, T cells into DN3/4 stage "thymocytes"). Apoptosis sensitivity will further be tested ex vivo from mice injected with progenitor cells (by injecting irradiated mice with progenitors, fully mature lymphocytes can be obtained). The expression of BH3-only proteins during differentiation towards B and T cells in vitro will further be measured. Through the assessment of complex formation with anti-apoptotic Bcl-2 proteins, open questions of the molecular function of BH3-only proteins will be addressed. Lastly, by measuring competitive productivity of differentiating, gene-deficient cells during injection of two genetically different sets of progenitor cells into mice (such as Bim- plus Bim/Noxa-deficient) we will assess potential survival advantages upon loss of BH3-only proteins during differentiation of myeloid and lymphoid cells in vivo. We believe that with this approach we will be able to obtain substantial new information on BH3-only proteins and their roles in differentiation and apoptosis sensitivity of mouse haematopoietic cells.

线粒体凋亡是造血细胞分化和存活过程中的重要调控机制。线粒体凋亡受Bcl-2蛋白家族控制;在该家族中，8种仅BH3蛋白是凋亡的启动子，其通过其表达水平和可能的活化调节凋亡。在免疫细胞中，Bim是最突出和最重要的仅含BH3的蛋白质，但许多其他仅含BH3的蛋白质（特别是Puma，Bmf和Noxa）似乎起着不完全理解的作用。我们已经了解了很多关于免疫细胞中线粒体凋亡的信息，特别是从基因缺陷小鼠模型中。然而，特别是由于在获得必要的细胞材料方面的限制，进一步的进展缓慢。我们建立了一种小鼠造血祖细胞体外扩增、体内外分化为髓系细胞和淋巴细胞的细胞模型。我们已经建立了这样的祖细胞从小鼠双缺乏BH3-唯一的蛋白（Bim/Puma，Bim/Bmf，Bim/Noxa），并发现有趣的凋亡缺陷在某些情况下。我们在这里建议使用这些细胞，并建立额外的细胞系携带三重缺陷的BH3-唯一的蛋白质，以分析的贡献BH3-唯一的蛋白质的发展，维护和凋亡的敏感性，特别是在B和T淋巴细胞。在体外，将测试祖细胞以及各种基因型的"B细胞"和"T细胞"的凋亡应答（B细胞可以在体外从祖细胞分化为IgM阳性细胞，T细胞分化为DN 3/4期"胸腺细胞"）。细胞凋亡敏感性将进一步从注射有祖细胞的小鼠中离体测试（通过用祖细胞注射经辐照的小鼠，可以获得完全成熟的淋巴细胞）。将进一步测量体外向B和T细胞分化期间仅BH 3蛋白的表达。通过评估与抗凋亡Bcl-2蛋白的复合物形成，将解决BH3-only蛋白的分子功能的开放问题。最后，通过测量在将两组遗传上不同的祖细胞注射到小鼠（例如Bim + Bim/Noxa缺陷型）期间分化的基因缺陷型细胞的竞争生产率，我们将评估在骨髓和淋巴样细胞体内分化期间仅BH 3蛋白质损失时的潜在存活优势。我们相信，通过这种方法，我们将能够获得大量的新信息BH3-only蛋白及其在小鼠造血细胞分化和凋亡敏感性中的作用。