The extracellular domain of human delta-like-1 expressed and purified from CHO cells promotes expansion of hematopoietic progenitor cells / 中国实验血液学杂志
Journal of Experimental Hematology
;
(6): 222-226, 2003.
Article
in Chinese
| WPRIM
| ID: wpr-355678
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Pharmacology
/
Physiology
/
Transcription Factors
/
Binding Sites
/
Recombinant Proteins
/
RNA
/
Hematopoietic Stem Cells
/
Glycoproteins
/
Transfection
/
Gene Expression
Limits:
Animals
/
Humans
Language:
Chinese
Journal:
Journal of Experimental Hematology
Year:
2003
Type:
Article
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