The protective effect of PEP-1-SOD1 preconditioning on hypoxia/reoxygenation injury in cultured human umbilical vein endothelial cells / 中华心血管病杂志
Chinese Journal of Cardiology
;
(12): 750-756, 2007.
Article
Dans Chinois
| WPRIM
| ID: wpr-307207
ABSTRACT
<p><b>OBJECTIVE</b>To construct prokaryotic expression vector of pET15b-PEP-1-SOD1 and investigate whether PEP-1-SOD1 fusion protein could be transduced into human umbilical vein endothelial cells (HUVECs) and the effects on hypoxia/reoxygenation injury.</p><p><b>METHODS</b>The recombinant plasmids pET15b-SOD1 and pET15b-PEP-1-SOD1 were constructed and transformed into E. coli BL21 (DE3) to express SOD1 and PEP-1-SOD1 with an N-terminal His-tag. The purified SOD1 and PEP-1-SOD1 were incubated with HUVECs and the viability (MTT assay) and the release of lactate dehydrogenase (LDH) in culture medium were determined in the hypoxia/reoxygenation injury model. The morphological changes were observed under an inverted phase contrast microscope. The content of malondialdehyde (MDA) in HUVECs was also determined with the method of thiobarbituric acid.</p><p><b>RESULTS</b>PEP-1-SOD1 fusion protein could be transduced into cultured HUVECs in a time- and dose-dependent manner. The intracellular enzymatic activity of PEP-1-SOD1 after 30 min incubation with HUVECs was significantly higher than control group (60.88 U/ml +/- 6.73 U/ml vs. 41.06 U/ml +/- 4.19 U/ml, P < 0.01). The transduced PEP-1-SOD1 protein was enzymatically stable for 24 h within cells. After hypoxia/reoxygenation injury, control HUVECs shrunk, became round-shaped and intercellular space increased, while these morphological changes were not observed in PEP-1-SOD1 transduced HUVECs. PEP-1-SOD1 transduction also markedly increased the viability, decreased LDH leakage into culture media and reduced the content of MDA post hypoxia/reoxygenation.</p><p><b>CONCLUSIONS</b>PEP-1-SOD1 fusion protein could be efficiently transduced into HUVECs in a natively active form, and the delivered enzymatically active PEP-1-SOD1 exhibits cellular protection against hypoxia/reoxygenation injury in HUVECs. The transduction of SOD1 mediated by cell-penetrating peptide, PEP-1, provides a basis for further research on the prevention of ischemia/reperfusion injury in vivo.</p>
Texte intégral:
Disponible
Indice:
WPRIM (Pacifique occidental)
Sujet Principal:
Peptides
/
Superoxide dismutase
/
Transduction génétique
/
Veines ombilicales
/
Protéines de fusion recombinantes
/
Lésion d'ischémie-reperfusion
/
Hypoxie cellulaire
/
Cellules cultivées
/
Mercaptamine
/
Biologie cellulaire
Limites du sujet:
Humains
langue:
Chinois
Texte intégral:
Chinese Journal of Cardiology
Année:
2007
Type:
Article
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