Mechanism of Differentiation and Proliferation of Granulocytes and Macrophages

粒细胞和巨噬细胞的分化和增殖机制

• 批准号: 59480137
• 负责人: NAGATA Shigekazu
• 金额: $ 4.03万
• 依托单位: Institute of Medical Science, University of Tokyo,
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1984
• 资助国家: 日本
• 起止时间: 1984 至 1986
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-59480137/
• 关键词: hemopoiesis; granulocyte colony stimulating factor; interferon- <gamma>; cDNA chromosomal gene; gene engineering; bovine papilloma virus; ミエロペルオキシダーゼ; 遺伝子工学

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

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) 等多种蛋白质因子的调节。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，会特异性诱导一些酶，例如髓过氧化物酶（MPO）。在这个项目中，我们分离了人 G-CSF 和 MPO 的 cDNA。从用组成型产生G-CSF的人鳞状细胞癌CHU-2细胞条件化的培养基中纯化人G-CSF，并测定G-CSF的部分氨基酸序列。通过使用寡核苷酸作为探针，分离出两种不同的人G-CSF cDNA，每种编码207或204个氨基酸。然后，通过使用人G-CSF cDNA作为探针，分离出鼠G-CSF的cDNA和G-CSF的染色体基因。人类和小鼠基因组中存在一个 G-CSF 基因（-2.5 kb 长）。仅在人类基因中，两个剪接供体位点串联排列在第二个内含子的 5' 端，这表明人类 G-CSF 的两种不同 mRNA 是通过选择性剪接产生的。人和鼠G-CSF在核苷酸和氨基酸序列水平上分别具有69.3%和72.6%的同源性。人MPO的cDNA是从HL-60细胞的cDNA文库中分离出来的。从cDNA阐明的蛋白质结构表明，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，并导入小鼠C127I细胞使用 牛乳头状瘤病毒作为载体。一些转化体可以在低血清培养基中非常有效地产生IFN-或G-CSF（1-20mg/1），并且每种蛋白质均被纯化至均质。它们在物理化学上与天然蛋白质没有区别。当重组G-CSF皮下注射到小鼠体内时，观察到显着的粒细胞生成和脾肿大。较少的

项目成果

Kazuhiro Morishita: Journal of Biological Chemistry. 262. (1987)

森下和宏：生物化学杂志。

Masayuki Tsuchiya: The EMBO Journal. 6. (1987)

土屋雅之：EMBO 杂志。

Kazuhiro Morishita: "Molecular Cloning and Characterization of cDNA for Human Myeloperoxidase" Journal of Biological Chemistry. 262. (1987)

Kazuhiro Morishita：“人髓过氧化物酶 cDNA 的分子克隆和表征”生物化学杂志。

Shigekazu Nagata: The EMBO Journal. 5. 575-581 (1986)

Shigekazu Nagata：EMBO 杂志。

Masayuki Tsuchiya: "Isolation and Characterization of the cDNA for Murine Granulocyte Colony Stimulating Factor" Proc. Natl. Acad. Sci. USA. 83. 7633-7637 (1986)

Masayuki Tsuchiya：“鼠粒细胞集落刺激因子 cDNA 的分离和表征”Proc。