Developing a system for regulating expression of human hepatocyte growth factor using tetracycline in NRK52E cells

Urol Int. 2010;85(2):228-36. doi: 10.1159/000314956. Epub 2010 Aug 10.

Abstract

Introduction: Hepatocyte growth factor (HGF) is a target of gene therapy for renal fibrosis. The aim of this study was to establish a human HGF gene expression system that is regulated by tetracycline (Tet) in normal rat kidney tubular epithelial cells (NRK52E cells).

Materials and methods: The plasmids pTet-on, pBI-L-HGF and pTK-Hyg were transfected sequentially into NRK52E cells using Lipofectamine 2000. The expression of HGF gene was measured, and the activity of expressed HGF was detected.

Results: A clone of pBI-L-HGF/NRK52E cells showing strong reaction to doxycycline (Dox) was selected using a luciferase reporter assay system. The expression of both HGF mRNA and protein was significantly higher (both p < 0.01) in the Dox group than that in the control group. Furthermore, the bioactivity of expressed HGF was confirmed in the assay.

Conclusions: A Tet-regulated human HGF gene expression system in NRK52E cells has been established. This cell line may prove useful for gene therapy against renal fibrosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Doxorubicin / pharmacology
  • Epithelial Cells / drug effects*
  • Epithelial Cells / metabolism
  • Epithelial-Mesenchymal Transition / drug effects
  • Gene Expression Regulation / drug effects
  • Hepatocyte Growth Factor / biosynthesis*
  • Hepatocyte Growth Factor / genetics
  • Humans
  • Kidney Tubules / drug effects*
  • Kidney Tubules / metabolism
  • RNA, Messenger / metabolism
  • Rats
  • Tetracycline / pharmacology*
  • Transfection
  • Transforming Growth Factor beta1 / metabolism
  • Up-Regulation

Substances

  • HGF protein, human
  • RNA, Messenger
  • Transforming Growth Factor beta1
  • Hepatocyte Growth Factor
  • Doxorubicin
  • Tetracycline