Isolation and Characterization of a Serum Component that Regulates Erythropoiesis

红细胞的寿命是有限的；哺乳动物一生都要制造红细胞。成熟红细胞的募集是通过未分化的造血祖细胞的增殖和分化实现的。促红细胞生成素(EPO)是肾脏产生的一种糖蛋白，是这些过程的主要生理刺激因子。当给予大鼠蛋白质剥夺死亡时，红细胞生成率受到损害。我们已经证明，这种损害是由于EPO合成的突然停止，似乎是在饮食中蛋白质被剥夺后立即发生的。本课题的目的是阐明EPO合成受控于蛋白质摄入的机制。(1)制备识别EPO不同表位的单抗，建立高灵敏度、高准确度、高可行性的EPO检测方法。建立的方法是夹心式酶联免疫分析法，在几个小时内可以检测到低至5mU/ml的EPO。(2)负责产生EPO的肾脏细胞尚不清楚。用该单抗对失血诱导产生EPO的大鼠肾脏进行染色。染色细胞的显微镜观察表明，肾小管周内皮细胞最有可能产生EPO。(3)为建立体外培养产生EPO的肾细胞的体系，用胶原酶破坏贫血大鼠肾皮质，制成单细胞悬液。培养细胞上清液中可检测到EPO的产生。对产生EPO的细胞进行了浓缩、纯化和鉴定，以便我们可以测试去蛋白饮食或蛋白质供应饮食的大鼠血清对培养细胞产生EPO的影响。

Erythrocytes have a limited life span ; mammals have to produce erythrocytes for their whole life. Recruitment of matured erythrocytes is achieved by proliferation and differentiation of undifferentiated hematopoietic precursors. Erythropoietin (EPO), a glycoprotein produced by kidney, is the major physiological stimulator of these processes. When protein-deprived died was given to rats, the rate of erythropoiesis is impaired. We have shown that this impairment is due to abrupt cessation of EPO synthesis that appears to occur immediately after deprivation of protein in the diet. The purpose of this project is to elucidate the mechanism by which the EPO synthesis is under control of protein intake.(1) To establish the method of EPO measurement with high sensitivity, high accuracy, and feasibility, monoclonal antibodies that recognize different epitopes of EPO have been prepared. The established method, a sandwich-type enzyme-linked immunoassay, measures EPO as low as 5 mU/ml within several hours. (2) The Kidney cells responsible for EPO production have been unclear. By the use of the monoclonal antibody, we stained the kidney of rat in which EPO production is induced by bleeding. The microscopic observation of the stained cells suggests that the peritubular endothelial cells are most likely candidate for production of EPO. (3) To develop the in vitro culture system of the kidney cells producing EPO, the kidney cortex of the anemic rat was disrupted by collagenase to yield single cell suspension. The EPO production was detected in the supernatant of the cultured cells. Concentration, purification, and identification of the cells producing EPO have being attempted so that we can test effect of the sera from the rats to which either protein-deprived or protein-supplied diet is given on EPO production of the cultured cells.