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Attenuated acute liver injury in mice by naked hepatocyte growth factor gene transfer into skeletal muscle with electroporation
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  1. F Xue1,
  2. T Takahara1,
  3. Y Yata1,
  4. M Minemura1,
  5. C Y Morioka1,
  6. S Takahara2,
  7. E Yamato3,
  8. K Dono4,
  9. A Watanabe1
  1. 1Third Department of Internal Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan
  2. 2Department of Urology, Osaka University Medical School, Osaka, Japan
  3. 3Department of Nutrition and Physiological Chemistry, Osaka University Medical School, Osaka, Japan
  4. 4Department of Surgery and Clinical Oncology, Osaka University Medical School, Osaka, Japan
  1. Correspondence to:
    Dr T Takahara, Third Department of Internal Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama, 930-0194 Japan;
    terutaka-tym{at}umin.ac.jp

Abstract

Background: Hepatocyte growth factor (HGF) plays an essential role in hepatic development and regeneration, and shows proliferative and antiapoptotic activity in hepatocytes.

Aims: To establish an effective new method for HGF gene transfer in vivo and to investigate its effects in acute experimental liver injury.

Animals: Eight week old female mice were used.

Methods: Rat HGF gene in a modified pKSCX plasmid was transferred to the tibialis anterior muscle by electroporation using a pulse generator. Four days later, plasma HGF concentrations were determined by enzyme linked immunosorbent assay every two days for three weeks. To confirm the efficacy of electroporation, a plasmid bearing green fluorescence protein (GFP) was transferred similarly. Four days after electroporation, carbon tetrachloride (CCl4) was administered to mice to induce acute liver injury. Plasma alanine aminotransferase (ALT) activity was measured. Hepatic apoptosis was assessed by Hoechst 33258 staining and the TUNEL method.

Results: Fluorescence microscopy showed strong green fluorescence where the GFP gene had been transferred into muscle. In mice given the HGF gene, HGF in plasma was increased up to fourfold from pretreatment amounts, peaking 6–9 days after electroporation and quickly decreasing within three weeks. Compared with the group without HGF transfer, the percentage of apoptotic hepatocytes after CCl4 intoxication was significantly lower, as was ALT activity. In addition, ALT activity normalised more rapidly in the HGF gene transfer group.

Conclusions: Naked DNA injection and transfer by electroporation efficiently brings about HGF expression in vivo, which can attenuate acute liver injury.

  • hepatocyte growth factor
  • gene therapy
  • acute liver injury
  • electroporation
  • HGF, hepatocyte growth factor
  • GFP, green fluorescence protein
  • ALT, alanine aminotransferase
  • ELISA, enzyme linked immunosorbent assay
  • CCl4, carbon tetrachloride
  • TdT, terminal deoxynucleotidyl transferase
  • TUNEL, TdT mediated dUTP-X nick end labelling
  • ABC, avidin-biotin complex
  • DAB, diaminobenzidine
  • PBS, phosphate buffered saline
  • PCNA, proliferative cell nuclear antigen
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