Abstract
Currently, there is a lack of systems for studying the role of hepatitis B viral proteins, such as HBeAg and HBcAg, on liver injury. It is necessary to develop an original tool in order to clarify the role of these viral proteins in hepatic stellate cell activation, and to understand the molecular mechanisms of liver injury. HepaRG are the most reliable hepatocyte-like cells for studying liver functions or disorders. In this paper, we demonstrate that the transduction of differentiated HepaRG (dHepaRG) cells can be performed successfully using lentiviral particles. The production of a functional Green Fluorescent Protein (GFP) assessed by Fluorescence Activated Cell Sorting and fluorescence microscopy is up to 16% of GFP positive cells using a multiplicity of infection (MOI) of 2.4. We demonstrate that this technology can allow the stable expression of GFP during the long lifecycle of the cell (up to four weeks after the cell’s passage). With this innovative tool, we aim to express viral proteins such as HBeAg or HBcAg in dHepaRG cells. The preliminary results of this work shows that HBeAg can be efficiently produced in dHepaRG cells and that increased MOI allows a better production of this protein. Our future objective will be to study the role of HBc and HBe proteins on the induction of hepatic fibrosis.
Highlights
There is a lack of systems for studying the role of hepatitis B viral proteins, such as HBeAg and HBcAg, on liver injury
Several cell types are implicated in the pathogenesis of liver fibrosis but hepatic stellate cell (HSC) activation is substantial in liver fibrosis[1]
Progenitor HepaRG cells can be differentiated in hepatocyte-like cells after exposure to DMSO and hydrocortisone, two well-known differentiation inducers4. differentiated HepaRG (dHepaRG) cells, showing a typical aspect of hepatocytes clustered in small colonies, offer similar molecular characteristics to Primary human hepatocytes (PHH), including morphology, nuclear receptors, expression of key metabolic enzymes, and drug transporters[4,5]
Summary
There is a lack of systems for studying the role of hepatitis B viral proteins, such as HBeAg and HBcAg, on liver injury. The production of a functional Green Fluorescent Protein (GFP) assessed by Fluorescence Activated Cell Sorting and fluorescence microscopy is up to 16% of GFP positive cells using a multiplicity of infection (MOI) of 2.4 We demonstrate that this technology can allow the stable expression of GFP during the long lifecycle of the cell (up to four weeks after the cell’s passage). After the transduction of pHepaRG, cell differentiation with DMSO, another treatment was performed in order to overexpress a receptor, or to silence a mitochondrial component[8,9,10] Another recent study used lentiviral technology to transduce pHepaRG and to express a Farnesoid receptor enabling study of the impact of this protein expression on the infection, replication and persistence of HBV11. Low or undetectable amounts of mRNAs were observed in proliferating HepaRG cells, whereas at confluence for one month, albumin and aldolase B mRNAs became highly expressed[4]
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