[The study on the construction of expression plasmid containing rHO-1 cDNA, analysis of rHO-1 activity and function of anti-apoptosis in HUVEC].
Xenograft rejection are due to the activation of endothelial cells and the expression of a series of proinflammatory genes encoding adhesion molecules, cytokines/chemokines and procoagulant molecules,leading to endothelial cell injury and apoptosis. HO-1 which acts as a cytoprotective gene can suppress a variety of inflammatory responses to prevent graft rejection. In this study, The plasmid containing HO-1 cDNA was constructed and transfected into human vascular endothelial cells (HUVEC) for expression using DOTAP lipsomal transfection reagents. Cells were homogenized in cell culture lysis reagent (CCLR) and the activity of HO-1 was measured. The apoptosis of HUVEC induced by tumor necrosis factor (TNF)-alpha was analyzed by flow cytometry. Meanwhile, Heme and Sn-protoporphyrin (SnPP) were added respectively to evaluate the apoptosis rate of HUVEC. The results showed that the expression of HO-1 gene can be significantly up-regulated in HUVEC transfected with pcDNA3HO1. The apoptosis rate of cells treated with Heme was less than 20% but significantly increased when cells treated with SnPP, the maximum arrived at 95%. The apoptosis rate in heme induced cells was 5-20 times lower than that in SnPP inhibited cells. The apoptosis rate was a negative relation to HO-1 expression. HO-1 protein can inhibit apoptosis in HUVEC. The results provide evidence to support the essential role of HO-1 in the cytoprotective function.
Xia
Zhen Wei
ZW
Department of Pediatrics, Rui Jin Hospital, Shanghai Second Medical University, Shanghai. xzw63@hotmail.com
Qi
Qi
Q
Zhang
Xue Hong
XH
Shen
Qing Xiang
QX
Li
Yun Zhu
YZ
Yu
Shan Chang
SC
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D015153:Blotting, Western; D018076:DNA, Complementary / Q000235:genetics*; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D006418:Heme / Q000494:pharmacology; D051547:Heme Oxygenase-1 / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D006801:Humans; D008665:Metalloporphyrins / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics*; D051381:Rats; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D018076:DNA, Complementary; D008665:Metalloporphyrins; D014409:Tumor Necrosis Factor-alpha; D006418:Heme; D051547:Heme Oxygenase-1