[The extracellular domain of human delta-like-1 expressed and purified from CHO cells promotes expansion of hematopoietic progenitor cells].
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.
Lu
Zhuo-Zhuang
ZZ
Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850, China.
Wu
Chu-Tse
CT
Liu
Hong-Jun
HJ
Zhang
Qun-Wei
QW
Jia
Xiang-Xu
XX
Wang
Li-Sheng
LS
D000818:Animals; D018952:Antigens, CD34 / Q000276:immunology; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D003114:Colony-Forming Units Assay; D006224:Cricetinae; D016228:Endothelial Growth Factors / Q000494:pharmacology; D005312:Fetal Blood / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006023:Glycoproteins / Q000235:genetics* / Q000494:pharmacology / Q000502:physiology; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology; D007377:Interleukin-3 / Q000494:pharmacology; D008222:Lymphokines / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics; D012313:RNA / Q000235:genetics / Q000378:metabolism; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019089:Stem Cell Factor / Q000494:pharmacology; D014157:Transcription Factors; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
D018952:Antigens, CD34; D016228:Endothelial Growth Factors; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D007377:Interleukin-3; D008222:Lymphokines; D008565:Membrane Proteins; C497761:NOTCH1 protein, human; D051881:Receptor, Notch1; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D019089:Stem Cell Factor; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; D012313:RNA