Stem cell growth factor: isolation of the cDNA, analysis on the gene expression, molecular characterization and in vitro proliferative activity of a recombmant preparation on primitive hematopoietic progenitor cells

干细胞生长因子：重组制剂对原始造血祖细胞的cDNA分离、基因表达分析、分子表征和体外增殖活性

• 批准号: 10557094
• 负责人: HIRAOKA Atsunobu
• 金额: $ 7.81万
• 依托单位: Osaka Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10557094/
• 关键词: SCGF; stem cell growth factor; recombinant gene; hematopoietic activity; bone marrow cells; CD34; C-type lectin; stroma; 活性; 受容体; 抗体; 造血因子; 遺伝子組換え; 造血活性; 骨髄CD34^+Lin^-細胞; 抗SCGF抗体; ラベル化SCGF; In situ hybridization; 骨・軟骨組織; 骨髄細胞; 赤芽球; 顆粒

On an attempt to realize diagnostic and therapeutic application of stem cell growth factor (SCGF) in hematology we studied molecular characterization, the gene expression and hematopoietic activities of a recombmant human (rh) SCGF. A full-length cDNA for human, mouse and rat SCGF was cloned using bone marrow cells, MC3T3-G2/PA6 preadipocytes and ROS-17/2.8-5 osteosarcoma cells, respectively, SCGF was a new member of a C-type lectin superfamily and most homologous with tetranectin. A 21aa signal peptide was removed to give rise to mature SCGF with MW 40 kDa. SCGF was not N-glycosylated, but O-glycosylated with sialic acid. Leucine zipper-like sequence could induce SCGF to form a ternary α-helical coiled-coil structure. Northern and in situ hybridization analysis showed specific expression of SCGF mRNA in the spleen, fetal liver and skeletal tissues, particularly in bone marrow cells, periosteum and proliferating chondrocytes, but little expression in resting or hypertrophic chondrocyte … More s, indicating that SCGF affected osteogenesis. Fluorescent in situ hybridization mapped human and mouse SCGF genes to chromosome 19q13.3 and 7B3-B5, respectively, where flt3 ligand and IL-11 genes were clustered. CHO cells were stably transformed with an expression vector PAGE-SCGF, and an endotoxin-free rhSCGF was purified from the culture supernatant. Anti-SCGF monoclonal antibodies were obtained by immunizing rats with a synthetic oligopeptide. For human bone marrow CD34+Lin- cells rhSCGF promoted growth of erythroid and granulocyte/macrophage colonies in the culture with erythropoietin and GM-CSF or G-CSF, respectively, and was synergistic with IL-3 and flt3 ligand to enhance GM colony-growth.SCGF is selectively produced by osseous and hematopoietic stromal cells and can mediate their proliferative activity on hematopoietic stem cells. For further characterization of SCGF, activities on other cells, receptor, knockout mice and clinical aspects should be studied, and SCGF is surely expected to build a new stem cell biology paradigm. Less

为了实现干细胞生长因子（SCGF）在血液学诊断和治疗中的应用，我们研究了重组人（rh）SCGF的分子特性、基因表达和造血活性。用骨髓细胞、MC 3 T3-G2/PA 6前脂肪细胞和ROS-17/2.8-5骨肉瘤细胞分别克隆了人、小鼠和大鼠SCGF的全长cDNA。去除21 aa信号肽，得到分子量为40 kDa的成熟SCGF。SCGF未被唾液酸N-糖基化，而是被唾液酸O-糖基化。亮氨酸拉链样序列可诱导SCGF形成三元α螺旋卷曲螺旋结构。北方杂交和原位杂交结果显示，SCGF mRNA在脾脏、胎肝和骨骼组织中特异性表达，尤其在骨髓细胞、骨膜和增殖软骨细胞中表达更强，而在静止和肥大软骨细胞中几乎不表达 ...更多信息 s，表明SCGF影响成骨。荧光原位杂交将人和小鼠SCGF基因分别定位于染色体19q13.3和7 B3-B5，其中flt 3配体和IL-11基因聚集。用表达载体PAGE-SCGF稳定转化CHO细胞，并从培养上清液中纯化无内毒素的rhSCGF。用合成的寡肽免疫大鼠，获得抗SCGF单克隆抗体。对于人骨髓CD_（34）~+Lin-细胞，rhSCGF分别与促红细胞生成素（EPO）、GM-CSF或G-CSF共同培养，促进红系和粒/巨噬细胞集落的生长，并与IL-3和flt 3配体协同促进GM集落的生长。对SCGF的进一步研究还需从其他细胞、受体、基因敲除小鼠和临床等方面进行，并有望建立一种新的干细胞生物学模式。少

项目成果

平岡篤信: "Stem cell growth factor : in situ hybridization analysis on the gene expression, molecular characterization and in vitro proliferative activity of a recombinant..."The Hematology Journal. 2・5. 307-315 (2001)

Atsunobu Hiraoka：“干细胞生长因子：重组体的基因表达、分子特征和体外增殖活性的原位杂交分析……”《血液学杂志》2·5（2001）。

美王宏之: "Isolation and characterization of a cDNA for human, mouse, and rat full-length stem cell growth factor, a new member of C-type lectin superfamily"Biochemical and Biophysical Research Communications. 249・1. 124-130 (1998)

Hiroyuki Mioh：“人、小鼠和大鼠全长干细胞生长因子（C 型凝集素超家族的新成员）的 cDNA 的分离和表征”《生物化学和生物物理研究通讯》（1998 年）。 ）

平岡篤信: "Stem cell growth factor : In situ hybridization analysis on gene expression, molecular characterization and in vitro proliferative activity of a recombinant preparation on primitive hematopoietic progenitor cells"The Hematology Journal. 2.(予定). (20

Atsunobu Hiraoka：“干细胞生长因子：原始造血祖细胞重组制剂的基因表达、分子特征和体外增殖活性的原位杂交分析”《血液学杂志》2。（计划中）。

Hiraoka, A., Yano, K., Kagami, N., Takeshige, K., Mio, H., Anazawa, H. and Sugimoto, S.: "Stem cell growth factor: in situ hybridization analysis on the gene expression, molecular characterization and in vitro proliferative activity of a recombinant prepa

Hiraoka, A.、Yano, K.、Kagami, N.、Takeshige, K.、Mio, H.、Anazawa, H. 和 Sugimoto, S.：“干细胞生长因子：基因表达的原位杂交分析，

Mio, H., Kagami, N., Yokokawa, S., Kawai, H., Nakagawa, S., Takeuchi, K., Sekine, S. and Hiraoka, A.: "Isolation and characterization of a cDNA for human, mouse, and rat full-length stem cell growth factor, a new member of C-type lectin superfamily"Bioche

Mio, H.、Kagami, N.、Yokokawa, S.、Kawai, H.、Nakakawa, S.、Takeuchi, K.、Sekine, S. 和 Hiraoka, A.：“人类 cDNA 的分离和表征，