[The stable expression of human tissue-type plasminogen activator gene mediated by lipofectamine in human vein endothelial cell line cells].
We have established a human umbilical vein endothelial cell (HUVEC) line monoclonal cells with the stable expression of human tissue-type plasminogen activator (t-PA) gene to provide a basis for further study on the vascular tissue engineering. Recombinant plasmid pcDNA3. 1-Myc-His B (-)/t-PA was constructed by insertion of t-PAcDNA originated from PBS/t-PA into eukaryotic expression vector pcDNA3. 1-Myc-His B(-) and transfected into hUVEC line cells mediated by lipofectamine. The positive clones were obtained by the screen of G418. The transcription and expression of t-PA gene were investigated by RT-PCR and Western blotting respectively. The t-PA activity was measured by chromogenic substrate assay. The positive clone cells which transcripted the mRNA of t-PA gene was obtained by RT-PCR. Immunoreactive human t-PA of the medium was significantly increased in the group of transfected gene when compared with that in the controlled and transfected plasmid without t-PA gene group. The biological activity of the protein of the t-PA in the media was increased significantly in the positive clone cells with t-PA gene transfected. The HUVEC line monoclonal cells with the stable expression of t-PA gene was established successfully.
Liu
Xiaobin
X
Department of Cardiovascular Surgery, Guizhou Provincial Hospital, Guiyang 550002, China. liuxiaobin930@yahoo.com.cn
Zhang
Kailun
K
Jiang
Xionggang
X
Wang
Jianing
J
Huang
Yongzhang
Y
D002460:Cell Line; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism*; D006801:Humans; D008055:Lipids / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D023822:Tissue Engineering; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
D008055:Lipids; C086724:Lipofectamine; D012333:RNA, Messenger; D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator