Hematopoietic Circadian Rhythms

大多数生物系统显示昼夜节律可能是由下丘脑的视交叉上核（SCN）信号调节的。造血节律已被确定为不同的祖细胞，骨髓DNA合成和各种造血活性细胞因子。我们现在已经确定了小鼠骨髓干细胞植入100只cGy治疗小鼠的显著生物节律。我们还建立了一个独特的，Lin- Ho（低）Rho（低）双外排小鼠干细胞分离，并确定了这些细胞的“R1”亚群适合这些昼夜节律研究。在目前的资助中，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，以及植入是否有季节性节律。我们还将评估移植节奏是否基于归巢节奏。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）的可移植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追踪模型。这将使我们能够监测干细胞分离和纯化过程后的节律。我们还将研究干细胞纯化后小鼠干细胞植入节律是否持续。我们将评估消融SCN是否会消除这些节律。最后，随着Per1-luc纯化大鼠干细胞的可用性，我们将使用免疫表型和阵列技术研究基因和蛋白质的表达。这些实验将采用遗传Ly-5.1到Ly-5.2或PVG RT7b到PVG RT7a（大鼠）移植于100 gy暴露的小鼠或大鼠，在光暗箱中调节啮齿动物，体外祖细胞克隆培养和氚化胸腺嘧啶自杀。将使用单克隆抗体、荧光原位杂交（FISH）和FACS分析来追踪供体细胞。转基因Pert-luc大鼠和小鼠将在弗吉尼亚大学获得，而阵列/信息学研究将在普林斯顿完成。

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.