title: Experimental study on establishing eukaryotic expression vector pcDNA3.1(+) TPA and its expressing activity in human skin fibroblists.
abstract: OBJECTIVE
  To establish a cell line stably expressing the tissue plasminogen activator (TPA) in human skin fibroblasts so as to develop the function analysis and gene therapy of TPA in ischemic heart diseases.
  
  
  METHODS
  Eukaryotic expression vector pcDNA3.1(+) TPA was constructed and transferred into human skin fibroblasts. After G418 selection, the exogenous expression and activity of TPA were observed subsequently by reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and chromogenic substrate assay.
  
  
  RESULTS
  Eukaryotic expression vector pcDNA3.1(+) TPA was expressed effectively in human skin fibroblasts. Quantitative ELISA showed that the expression of TPA protein of the experiment group was much higher than that of the control group (643.5 ng/10(6) cells per 24 hours vs. 19.2 ng/10(6) cells per 24 hours). And the chromogenic substrate assay showed that the exogenous TPA activity of the experimental group was also much higher than that of the control group (122.6 U/10(6) cells per 24 hours vs. 5.8 U/10(6) cells per 24 hours).
  
  
  CONCLUSIONS
  The exogenous TPA gene can be expressed effectively after pcDNA3.1(+)TPA was transferred into human skin fibroblasts, suggesting that the cell model will become an important tool in the further study of TPA function and gene therapy in ischemic heart diseases.
authors.affiliation: Department of Gerontology, Second Xiangya Hospital, Central South University, Changsha 410011, China.
authors.forename: Hong-bo
authors.initials: HB
authors.lastname: Deng
authors.forename: Yu
authors.initials: Y
authors.lastname: Yang
authors.forename: Jin-fu
authors.initials: JF
authors.lastname: Yang
authors.forename: Tao
authors.initials: T
authors.lastname: Tang
authors.forename: Wen-wu
authors.initials: WW
authors.lastname: Zhou
authors.forename: Jian
authors.initials: J
authors.lastname: Liu
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D018076:DNA, Complementary; D005057:Eukaryotic Cells / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D012867:Skin / Q000166:cytology*; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D010959:Tissue Plasminogen Activator