The extracellular domain of human delta-like-1 expressed and purified from CHO cells promotes expansion of hematopoietic progenitor cells.
- Author:
Zhuo-Zhuang LU
1
;
Chu-Tse WU
;
Hong-Jun LIU
;
Qun-Wei ZHANG
;
Xiang-Xu JIA
;
Li-Sheng WANG
Author Information
1. Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Antigens, CD34;
immunology;
Binding Sites;
genetics;
CHO Cells;
Cell Division;
drug effects;
physiology;
Colony-Forming Units Assay;
Cricetinae;
Endothelial Growth Factors;
pharmacology;
Fetal Blood;
cytology;
immunology;
metabolism;
Gene Expression;
Genetic Vectors;
genetics;
Glycoproteins;
genetics;
pharmacology;
physiology;
Hematopoietic Stem Cells;
cytology;
drug effects;
Humans;
Intercellular Signaling Peptides and Proteins;
pharmacology;
Interleukin-3;
pharmacology;
Lymphokines;
pharmacology;
Membrane Proteins;
genetics;
RNA;
genetics;
metabolism;
Receptor, Notch1;
Receptors, Cell Surface;
Recombinant Proteins;
isolation & purification;
pharmacology;
Reverse Transcriptase Polymerase Chain Reaction;
Stem Cell Factor;
pharmacology;
Transcription Factors;
Transfection;
Vascular Endothelial Growth Factor A;
Vascular Endothelial Growth Factors
- From:
Journal of Experimental Hematology
2003;11(3):222-226
- CountryChina
- Language:Chinese
-
Abstract:
Notch signal path plays important roles in the regulation of proliferation and differentiation of hematopoietic stem cells. An extracellular domain of human Delta-like-1 (hDll-1(ext)), one of Notch ligands, was cloned and expressed in CHO cells, and the effect of hDll-1(ext) on expansion of hematopoietic stem/progenitor cells was investigated in this study. Total RNA was isolated from human marrow mononuclear cells. hDll-1(ext) was amplified by RT-PCR and cloned to T vector, then the gene was sequenced and subcloned to pcDNA3.1/Myc-His(+)A expression vector. The constructed plasmid was transfected into CHO cells with lipofectin and the expression of secreted hDll-1(ext) in G418-resistant clones was assayed by Western blot. hDll-1(ext) high-expressed clone was cultured to collect supernatant. Fusion protein hDll-1(ext) was purified from the supernatant by immobilized metal affinity chromatography (IMAC). The results showed that expression of Notch-1 receptor was detected in cord blood-derived CD34(+) cells by RT-PCR. Human umbilical blood CD34(+) cells were cultured in serum-free medium containing SCF, IL-3, VEGF, and with or without purified hDll-1(ext) for 4 or 8 days. Effect of hDll-1(ext) on the expansion of progenitor cells was analyzed then by clonogenic assays. The number of CFU-Mix and HPP-CFC generated from the culture system containing hDll-1(ext) was 1.5 times of that from the control. In conclusion, the recombinant hDll-1(ext) promotes the expansion of primitive hematopoietic progenitors.
