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

线粒体凋亡是造血细胞分化和生命过程中的重要调控机制。线粒体凋亡受bcl -2家族蛋白调控；在这个家族中，8个BH3-only蛋白是细胞凋亡的启动因子，它们的表达水平和可能的激活调节细胞凋亡。在免疫细胞中，Bim是最突出和最重要的BH3-only蛋白，但许多其他BH3-only蛋白（尤其是Puma、Bmf和Noxa）似乎起着不完全清楚的作用。我们对免疫细胞中的线粒体凋亡，特别是基因缺陷小鼠模型，已经有了很多了解。然而，进一步的进展是缓慢的，特别是因为在获得必要的细胞材料方面的限制。我们建立了小鼠造血祖细胞体外扩增、体外和体内分化为骨髓细胞和淋巴细胞的细胞模型。我们已经从BH3-only蛋白双缺陷小鼠（Bim/Puma, Bim/Bmf, Bim/Noxa）中建立了这样的祖细胞，并在某些情况下发现了有趣的凋亡缺陷。我们建议利用这些细胞，并建立其他携带BH3-only蛋白三重缺陷的细胞系，以分析BH3-only蛋白对发育、维持和凋亡敏感性的贡献，特别是在B淋巴细胞和T淋巴细胞中。在体外，我们将检测祖细胞以及不同基因型的“B细胞”和“T细胞”的凋亡反应（B细胞可以从祖细胞体外分化为igm阳性细胞，T细胞可以分化为DN3/4期的“胸腺细胞”）。细胞凋亡敏感性将进一步在体外测试小鼠注射祖细胞（通过注射祖细胞照射小鼠，可以获得完全成熟的淋巴细胞）。BH3-only蛋白在体外向B细胞和T细胞分化过程中的表达将进一步测定。通过评估与抗凋亡Bcl-2蛋白的复合物形成，将解决BH3-only蛋白分子功能的开放性问题。最后，通过测量向小鼠注射两组基因不同的祖细胞（如Bim + Bim/Noxa-deficient）时分化的基因缺陷细胞的竞争生产力，我们将评估骨髓和淋巴细胞在体内分化过程中BH3-only蛋白丢失后的潜在生存优势。我们相信，通过这种方法，我们将能够获得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的大量新信息。

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.