Mechanism of Differentiation and Proliferation of Granulocytes and Macrophages

造血细胞的增殖、分化和活化受干扰素（ifn）和集落刺激因子（csf）等多种蛋白因子的调控。粒细胞集落刺激因子（G-CSF）是中性粒细胞的调节因子，在粒细胞分化过程中，一些酶如髓过氧化物酶（MPO）被特异性诱导。在这个项目中，我们分离了人G-CSF和MPO的cdna。从人鳞癌细胞组成性产生G-CSF的培养基中纯化人G-CSF，并测定其部分氨基酸序列。以寡核苷酸为探针，分离出两种不同的人G-CSF cdna，分别编码207或204个氨基酸。然后，以人G-CSF cDNA为探针，分离鼠G-CSF cDNA和G-CSF染色体基因。在人和小鼠基因组中，G-CSF有一个-2.5 kb长的单基因。只有在人类基因中，两个剪接供体位点在第2内含子的5'端串联排列，这表明两种不同的人G-CSF mrna是由另一种剪接产生的。人和鼠G-CSF在核苷酸和氨基酸序列水平上的同源性分别为69.3%和72.6%。从HL-60细胞cDNA文库中分离到人MPO cDNA。蛋白质结构分析表明，MPO蛋白作为Mr.84,000的前体合成，随后的修饰和切割产生重链（Mr. 55-60,000）和轻链（Mr. 15,000）。将人IFN- < γ >和G-CSF的cdna置于SV40早期启动子的控制下，以牛乳头瘤病毒为载体导入小鼠C127I细胞。一些转化子在低血清培养基中可以非常有效地产生IFN-或G-CSF (1-20 mg / 1)，并且每种蛋白都被纯化到均匀性。它们在物理化学上与天然蛋白质难以区分。重组G-CSF皮下注射小鼠后，观察到明显的颗粒生成和脾肿大。少

Proliferation, differentiation and activation of hematopoietic cells are regulated by several protein factors like interferons (IFNs) and colony stimulating factors (CSFs). Granulocyte colony stimulating factor (G-CSF) is a regulator for neutrophilic granulocytes, and during differentiation of granulocytes, some enzymes such as myeloperoxidase (MPO), are specifically induced. In this project, we have isolated cDNAs for human G-CSF and MPO. Human G-CSF was purified from the medium conditioned with human squamous carcinoma CHU-2 cells producing G-CSF constitutively, and the partial amino acid sequence of the G-CSF was determined. By using an oligonucleotide as probe, two different cDNAs for human G-CSF were isolated, each encoding 207 or 204 amino acids. Then, by using the human G-CSF cDNA as probe, cDNA for murine G-CSF and chromosomal genes for G-CSF were isolated. There is a single gene (-2.5 kb long) for G-CSF in human and mouse genome. Only in human gene, two splice donor sites are … More arranged in tandem at the 5' end of the 2nd intron, which suggests that two different mRNAs for human G-CSF are generated by an alternative splicing. Human and murine G-CSF have homologies of 69.3 % and 72.6 % on the nucleotide and amino acid sequence level, respectively.The cDNA for human MPO was isolated from the cDNA library of HL-60 cells. The protein structure elucidated from the cDNA indicated that the MPO protein is synthesized as a precursor of Mr.84,000, and subsequent modification and cleavage result in the heavy chain (Mr. 55-60,000) and the light chain (Mr. 15,000).The cDNAs for human IFN- <gamma> and G-CSF were placed under the control of SV40 early promoter, and introduced into mouse C127I cells using bovine papilloma virus as a vector. Some of the transformants could produce IFN- or G-CSF very efficiently ( 1-20 mg / 1 ) in a low serum medium, and each proteins was purified to homogeneity. They are indististiguishable physicochemically from the native proteins. When the recombinant G-CSF was subcutaneously administrated into mice, a remarkable granulopoiesis and splenomegaly were observed. Less