Hematopoietic Circadian Rhythms

大多数生物系统显示出昼夜节律，可能是由下丘脑的视交叉上核(SCN)发出的信号调节的。不同的造血祖细胞、骨髓DNA合成和各种造血活性细胞因子的造血学节律已被确定。我们现在已经确定了小鼠骨髓干细胞植入100cGy射线治疗的小鼠的显著生物节律。我们还建立了一个独特的，林和(低)Rho(低)双外排小鼠干细胞分离，并确定了这些细胞的“R1”亚群适合于这些昼夜节律研究。在目前的拨款中，我们计划评估外来因素的作用，短期(3周)和长期(6、9或12个月)的节律，以及是否有季节性的节律可供植入。我们还将评估植入节奏是否基于归位节奏。为了促进这些研究，并开始研究这些节律的分子和生化机制，我们将研究PER1-Luc转基因大鼠(和小鼠)的植入性和荧光素酶表达的时间节律。作为这些研究的前提，我们将建立大鼠干细胞纯化模型和大鼠骨髓移植跟踪模型。这将使我们能够监测干细胞分离和纯化程序后的节律。我们还将研究在干细胞纯化程序后，小鼠干细胞植入节律是否持续。我们将评估消融SCN是否会取消这些节律。最后，随着PER1-Luc纯化的大鼠干细胞的问世，我们将利用免疫表型和阵列技术来研究基因和蛋白的表达。这些实验将使用Ly-5.1到Ly-5.2或PVG RT7b到PVG RT7a(大鼠)的同源基因移植到100cGy射线照射的小鼠或大鼠，在明暗箱中进行啮齿动物的条件反射，体外祖细胞克隆培养和氚胸苷自杀。供体细胞将使用单抗、荧光原位杂交(FISH)和流式细胞仪分析进行追踪。转基因PERT-LUC大鼠和小鼠将在弗吉尼亚大学获得，阵列/信息学研究将在普林斯顿大学完成。

Most biologic systems show circadian rhythms probably regulated by signals from the suprachiasmatic nucleus (SCN) in the hypothalamus. Hematopoietic rhythms have been identified for different progenitor cells, marrow DNA synthesis, and various hematopoietically active cytokines. We have now identified prominent biologic rhythms for murine marrow stem cell engraftment into 100 cGy treated mice. We have also established a unique, Lin- Ho(low) Rho(low) double efflux murine stem cell separation, and identified the "R1" subpopulation of these cells as appropriate for these circadian studies. In the present grant, we plan to evaluate the role of extraneous factors, the rhythms of short-term (3 weeks) and longer-term (6, 9 or 12 months), and whether there are seasonal rhythms for engraftment. We will also evaluate whether the engraftment rhythm is based on a homing rhythm. In order to facilitate these studies and to begin to investigate molecular and biochemical mechanisms of these rhythms, we will study the temporal rhythms of engraftability and luciferase expression in Per1-luc transgenic rats (and mice). As a prerequisite for these studies, we will establish a rat stem cell purification model and a rat marrow transplantation-tracking model. This will enable us to monitor rhythms after stem cell separation and purification procedures. We will also study whether murine stem cell engraftment rhythms persist after a stem cell purification procedure. We will evaluate whether ablating the SCN abolishes these rhythms. Lastly, with availability of Per1-luc purified rat stem cells, we will study gene and protein expression using immunophenotyping and array technology. These experiments will employ congenic Ly-5.1 to Ly-5.2 or PVG RT7b to PVG RT7a (rats) transplants in 100 cGy exposed mice or rats, conditioning of rodents in light-dark boxes, in vitro progenitor clonal culture and tritiated thymidine suicide. Donor cells will be tracked using monoclonal antibodies, fluorescence in situ hybridization (FISH), and FACS analysis. Transgenic Pert-luc rats and mice will be available at University of Virginia, and the array/informatic studies done at Princeton.