Interleukin-10 gene modification attenuates hepatocyte activation of rat hepatic stellate cells in vitro.

Activation of hepatic stellate cells (HSCs) plays a key role in the progression of liver fibrosis. Interleukin-10 (IL-10), a potential anti-fibrosis cytokine, has an unfavorable pharmacokinetic profile, which limits its clinical applications. A liver-targeting gene delivery system may maintain a longer-lasting concentration in hepatic tissue with fewer side­effects in non-target tissues. In the present study, when delivered by asialoglycoprotein receptor-mediated liposomes, the IL-10 gene was highly expressed in BRL cells (a rat hepatocyte line) and attenuated the apoptosis of BRL cells induced by plasmid transfection. In a co-culture system, BRL cells demonstrated a marked ability to stimulate the proliferation of primary HSCs and their expression of α-SMA and procollagen type I. Following modification of the BRL cells with the IL-10 gene, this stimulation was attenuated and an accelerated apoptosis of the HSCs was induced. These results suggest that hepatocyte­targeting gene delivery may be an ideal technique for the IL-10 gene therapy of liver fibrosis, which requires further confirmation by in vivo studies.

[Construction of eukaryotic expression plasmid containing rat interleukin-10 gene and its expression in BRL cells in vitro].

AIM: To construct eukaryotic expression vector of rat IL-10 gene and observe its expression in hepatocyte cell line BRL. METHODS: Total RNA was extracted from rat peripheral blood mononuclear cells. The full length coding region of IL-10 was amplified by RT- nested PCR and cloned into eukaryotic expression vector pcDNA3.0. The recombinant plasmid was transfected into BRL cells with either liposome Transfast or asialoglycoprotein receptor mediated liposome PEIjet-gal respectively. The expression of IL-10 mRNA was detected with PCR and that of IL-10 secreted from BRL cells transfected by liposome PEIjet-gal was detected with ELISA. RESULTS: The recombinant plasmid was identified and confirmed with digestion of restriction endonuclease and DNA sequencing. Receptor mediated liposome PEIjet-gal exhibited significantly higher transfection efficiency than liposome Transfast and higher level secretory IL-10 expressed in BRL cells. CONCLUSION: The eukaryotic expression vector of IL-10 gene was successfully constructed. Asialoglycoprotein receptor-mediated liposome had high transfection efficiency on hepatocytes, suggesting that it could be a potential hepatocyte-targeting delivery system for IL-10 gene therapy.

[Targeted blockage of STAT5 by a decoy oligodeoxynucleotide inhibits the growth and proliferation of K562 cells].

OBJECTIVES: To investigate targeted blockage of BCR/ABL oncoprotein mediated cell transformation by STAT5 decoy oligodeoxynucleotide (ODN), its effect on the growth and proliferation inhibition of K562 cells and the related molecular mechanisms. METHODS: STAT5 decoy ODN, designed and synthesized in vitro, was transfected into K562 cells by cationic lipid. The cell growth curve and colony formation assay were used to reflect the growth and proliferation capacity of K562 cells, RT-PCR to detect the expression of three genes downstream STAT5. RESULTS: Confocal microscopy demonstrated that STAT5 decoy ODN was successfully transfected into K562 cells (95.2% positive cells). STAT5 decoy ODN inhibited the growth of K562 cells (inhibition rate 77.7%) and their colony formation capacity (Decoy ODN treated group 8.3% vs control group 35.7%, P < 0.05) after the treatment with STAT5 decoy ODN, the expressions of c-myc, bcl-X(L), cyclin D1 mRNA were down-regulated by 15.4%, 30.8%, 29.1%, respectively in the K562 cells. CONCLUSIONS: STAT5 decoy ODN inhibits the growth and proliferation of K562 cells. The mechanisms may be that decoy ODN blocks the transcriptional activation potent of STAT5 and down-regulates the expression of these tumor related genes downstream STAT5.