STEM CELLS AND AGING

描述(由申请人提供)：骨髓干细胞是衰老的决定性因素的假设将在早衰的小鼠模型中进行测试。首先，对野生型P53+/+和早衰P53+/m C57BL/6J小鼠的骨髓干细胞的数量和归巢能力进行体外和体内检测。然后，将测定野生型骨髓干细胞纠正p53+/m突变小鼠早衰的能力，以及野生型受者早衰p53+/m突变小鼠骨髓干细胞诱导早衰的能力。在具体目标1中，野生型P53+/+、P53+/-和P53+/m小鼠的干细胞数量将通过流式细胞术和体外克隆形成以及通过体外迁移和体内骨髓再繁殖确定的归巢来确定。本实验将2只GFP+雄性C57BI6 P53+/+小鼠的骨髓细胞移植到P53突变型(P53+/m)和野生型(P53+/+)雌性小鼠体内，观察其对衰老表型发育的影响。供体细胞对受体组织的贡献将通过绿色荧光蛋白和Y染色体的存在来确定。在这个模型中，如果衰老是由于骨髓干细胞对正常组织的替代能力降低，那么将正常小鼠的骨髓细胞移植到p53突变小鼠应该可以恢复正常的表型和正常的寿命。具体目的是将GFP+P53+/m雄性突变小鼠的3个骨髓细胞移植到照射后的正常野生型雌性小鼠和雌性突变小鼠体内，以确定衰老表型是否可以转移突变的骨髓干细胞。最后，将确定供体骨髓细胞与受体小鼠组织细胞融合的可能性，以及肿瘤中融合细胞的存在。这些实验的结果应该为成体干细胞最重要的假设特性之一提供关键证据。

DESCRIPTION (provided by applicant): The hypothesis that bone marrow stem cells are a determining factor in ageing will be tested in a mouse model of premature ageing. First, the number and homing capacity of bone marrow stem cells from wild type p53+/+ and premature ageing p53+/m C57BL/6J mice will be assayed in vitro and in vivo. Then, both the ability of wild-type bone marrow stem cells to correct premature ageing in p53+/m mutant mice, and the ability of bone marrow stem cells from premature ageing p53+/m mutant mice to induce premature ageing, in wild-type recipients will be determined. In Specific Aim 1, the number of stem cells in wild-type p53+/+, p53+/- and p53+/m mice will be determined by flow cytometry and by in vitro colony formation, and homing determined by in vitro migration and marrow repopulation in vivo. In Specific Aim 2 bone marrow cells from GFP+ male C57BI6 p53+/+ mice will be transplanted to p53 mutant (p53+/m) and to wild-type (p53 +/+) female mice and the effect on the development of the aging phenotype determined. The contribution of donor cells to recipient tissues will be identified by the presence of GFP and the Y-chromosome. If ageing is due to a decreased ability of bone marrow stem cells to contribute to replacement of normal tissues in this model, transplantation of bone marrow cells from normal mice to p53 mutant mice should restore normal phenotype and normal life span. In Specific Aim 3 bone marrow cells from GFP+ p53+/m male mutant mice will be transplanted to irradiated normal wild-type female mice and to female mutant mice to determine if the aging phenotype can be transferred mutant bone marrow stem cells. Finally the possibility of fusion of donor bone marrow cells with tissue cells of recipient mice and the presence of fused cells in tumors will be determined. The results of these experiments should provide critical proof for one of the most important hypothetical properties of adult stem cells.