Influence of hypoxia-inducible factor-1 alpha lentiviral vector on osteogenic gene expression of rabbit bone marrow mesenchymal stem cells
10.3969/j.issn.2095-4344.2014.01.010
- VernacularTitle:低氧诱导因子1α慢病毒载体转染骨髓间充质干细胞的成骨基因表达
- Author:
Jine ZHANG
;
Shuhong WANG
;
Xin ZHANG
;
Huayan GUO
;
Na GUO
;
Yuanliang HUANG
- Publication Type:Journal Article
- Keywords:
lentivirus;
mesenchymal stem cells;
bone marrow;
bone morphogenetic proteins;
viruses;
transfection
- From:
Chinese Journal of Tissue Engineering Research
2014;(1):57-62
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND:Osteogenesis is closely integrated with angiogenesis in bone formation and repair process, and hypoxia-inducible factor 1 alpha (HIF-1α) is considered to be the most important core transcription factor promoting angiogenesis gene regulation, which may promote the formation of blood vessels at hypoxia portion, and thus contribute to bone formation.
OBJECTIVE:To construct the Lenti-HIF-1α-IRES-EGFP (wild type) and Lenti-HIF-1α-IRES-EGFP (point mutant type) lentiviral eukaryotic expression vectors and to detect their impact on the osteogenic gene expression of rabbit bone marrow mesenchymal stem cells.
METHODS:The Lenti-HIF-1α-IRES-EGFP (wild type) and Lenti-HIF-1α-IRES-EGFP (point mutant type) lentiviral eukaryotic expression vectors were constructed according to the wild type human HIF-1αgene sequence and the determined restriction sites of human HIF-1αpoint mutant sequence. Rabbit bone marrow mesenchymal stem cells were transfected with the prepared Lenti-HIF-1α-IRES-EGFP (wild type) and Lenti-HIF-1α-IRES-EGFP (point mutant type) virus solution.
RESULTS AND CONCLUSION:Immunofluorescence microscopy observations indicated that the cells of Lenti-HIF-1α-IRES-EGFP (wild type) group and Lenti-HIF-1α-IRES-EGFP (point mutant type) group showed no obvious fluorescence on transfected 7 days, and two groups of cells showed a more obvious green fluorescent after transfection 14 days. Quantitative PCR analysis results showed that there were obvious HIF-1αand bonemorphogenetic protein 2 gene expressions on days 7 after transfection and the two genes stil showed highly expression levels after transfection 14 days. The two lentiviral eukaryotic expression vectors of Lenti-HIF-1α-IRES-EGFP (wild type) and Lenti-HIF-1α-IRES-EGFP (point mutant type) could be constructed according to the wild type human HIF-1αgene sequence and the determined restriction sites of human HIF-1αpoint mutant sequence;HIF-1αgene can promote the osteogenic gene expression of lentivirus-transfected rabbit bone marrow mesenchymal stem cells.
