The Effects Of Rat Hepatocytes Transfected With Interleukin-10 Gene On Primary Hepatic Stellate Cells

Aim:To investigate the effects of hepatocyte line and interleukin-10(IL-10) on the proliferation, activation and apoptosis of primary hepatic stellate cells(HSC).Method: The full length coding region of IL-10 was amplified by RT- nested PCR and cloned into eukaryotic expression vector pcDNA3.0. Then the recombinant plasmid pcDNA3.0-rIL-10 was identified with digestion of restriction endonuclease and DNA sequencing. The recombinant plasmid was transfected into hepatocyte line BRL cells with either liposome TransfastTM or asialoglycoprotein receptor mediated liposome PEIjet-gal respectively. The expression of IL-10 mRNA was detected by RT-PCR and that of IL-10 secreted from BRL cells transfected with liposome PEIjet-gal was detected by ELISA. MTT assay and flow cytometric analysis was used to detect proliferation and apoptosis of BRL cells transfected by plasmid pcDNA3.0 or pcDNA3.0-rIL-10. Both normal BRL cells and BRL cells transfected by plasmid pcDNA3.0 or pcDNA3.0-rIL-10 were coculture with primary HSC respectively. Phenotypic changes of HSC in each group were subsequently detected by phase contrast microscope, proliferation by MTT assay, apoptosis by TUNEL, expression ofα-SMA and procollagen type I by western blot. Cytokines differential expression of different groups BRL cells were analysed by using cytokines antibody arrays.Result: The recombinant plasmid pcDNA3.0-rIL-10 was successfully constructed, and transfected into BRL cells followed by higher level secretory IL-10 expression, which was subseqently showed to decrease apoptosis of BRL. Receptor mediated liposome PEIjet-gal exhibited significantly higher transfection efficiency than liposome TransfastTM. BRL cells significantly facilitated proliferation, apoptosis, contractllity, retinoid loss,α-SMA and procollagen type I expression of primary HSC. In contrast, IL-10 inhibited proliferation,α-SMA and I type collagen expression of primary HSC, and promoted apoptosis of HSC at the same time. Antibody arrays indicated that there was up-regulation of vascular endothelial growth factor(VEGF), monocyte chemoattractant protein-1(MCP-1), tissue inhibitor of metalloproteinase-1(TIMP-1) in the supernatant of BRL cells, and besides higher level of IL-10, there was lower level of VEGF in supernatant of BRL cells transfected by plasmid pcDNA3.0-rIL-10 than that of BRL cells transfected by plasmid pcDNA3.0.Conclusions: Asialoglycoprotein receptor-mediated liposome has high transfection efficiency on hepatocytes, suggesting that it could be a potential hepatocyte-targeting delivery system for IL-10 gene therepy. Hepatocytes stimulate proliferation and activation of HSC, which perhaps partly due to hepatocellular expression of VEGF, MCP-1 and TIMP-1. IL-10 gene-transfection leads to attenuation of this stimulation and acceleration of HSC apoptosis. The mechanism is that IL-10, in addition to its direct action to HSC, may also indirectly act to HSC by downregulating VEGF secreted from hepatocytes.