Mechanism of Differentiation and Proliferation of Granulocytes and Macrophages

造血细胞的增殖、分化和激活受多种蛋白质因子的调节，如干扰素(IFN)和集落刺激因子(CSF)。粒细胞集落刺激因子(G-CSF)是中性粒细胞的调节剂，在粒细胞分化过程中，髓过氧化物酶(MPO)等酶被特异性诱导。在本项目中，我们分离了人G-CSF和MPO的cDNA。从人鳞癌CHU-2细胞产生G-CSF的条件培养液中分离纯化出人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基因，分别编码207或204个氨基酸。然后，以人G-CSF基因为探针，分离出小鼠G-CSF基因和染色体G-CSF基因。人和小鼠基因组中只有一个G-CSF基因(-2.5kb)。只有在人类基因中，两个剪接供体位置是…更多的基因在第二内含子的5‘端串联排列，这表明人类G-CSF的两个不同的mRNAs是由另一种剪接产生的。人和小鼠G-CSF在核苷酸和氨基酸水平上的同源性分别为69.3%和72.6%。蛋白质结构分析表明，MPO蛋白是作为Mr 84,000的前体合成的，随后的修饰和切割产生了重链(Mr 55-60,000)和轻链(Mr 15,000)。将人干扰素和G-CSF的cDNA置于SV40早期启动子的控制下，并以牛乳头瘤病毒为载体导入小鼠C127I细胞。部分转化子能在低血清条件下高效生产干扰素或粒细胞集落刺激因子(1-20 mg/l)，并对每种蛋白进行了纯化。它们在物理化学上与天然蛋白质密不可分。将重组G-CSF皮下注射小鼠，可见明显的粒细胞生成和脾肿大。较少

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