[10, 11] In a previous study, we demonstrated that CCR5, but not CCR1, regulates the trafficking of immune cells into the liver under normal conditions. In addition, we also reported that in multidrug resistance 2 gene (Mdr2)-knockout (Mdr2-KO) mice, a strain that spontaneously develops chronic cholestatic hepatitis and fibrosis that is eventually followed by HCC,[13-15] the RANTES chemokine is highly expressed. RANTES is a ligand for both CCR1 and CCR5. Based on these observations, we propose, in this study, that the trafficking
of immune cells to the liver mediated by CCR5 is critical for the development of inflammation-induced HCC.[12, 17] To test this hypothesis,
we studied the role of CCR1 and CCR5 Selleckchem HDAC inhibitor in Mdr2-KO mice. Therefore, we generated two new strains Stem Cell Compound Library screening from the Mdr2-KO mouse, the Mdr2:CCR5 and the Mdr2:CCR1 double knockouts (DKOs), and set out to compare inflammation and tumorigenesis among these strains. Animal experiments were performed according to a protocol approved by the animal care committee of Hebrew University (Jerusalem, Israel). All animals were kept on a 12-hour light/dark cycle in a pathogen-free animal facility with free access to food and water. Wild-type (WT) C57Bl/6J and CCR5-deficient mice were purchased from The Jackson Laboratory (Bar Harbor, ME). CCR1-deficient mice were acquired from Taconic Farms (Germantown, NY). FVB.129P2-Abcb4tm1Bor (Mdr2-KO; The Jackson Laboratory) mice were kindly given to us by Dr. Daniel Goldenberg MCE公司 from the Goldyne Savad Institute of Gene Therapy Hadassah University Hospital (Jerusalem, Israel) and crossed into the C57Bl/6 genetic background for at least nine generations. Double-mutant Mdr2:CCR5 and Mdr2:CCR1 DKO mice were generated by crossing Mdr2-KO with either CCR5- or CCR1-deficient mice and their
progeny were identified by polymerase chain reaction (PCR) analysis (for primer sequences, see the Supporting Materials). At ages of 1, 3, and 16 months, mice were sacrificed by a lethal dose of isoflurane anesthesia and livers were excised and weighed. All mice were injected intraperioneally (IP) with bromodeoxyuridine (BrdU; Sigma-Aldrich, Rehovot, Israel) at 1 mg/mouse in 10 µL per 1 g of body weight 3 and 24 hours before sacrifice. Liver specimens were either fixed in 4% buffered formalin or snap-frozen in liquid nitrogen for further analysis. Blood samples were collected monthly from the age of 1 month until 6 months by tail vein bleeding. Levels of liver enzymes in sera, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase, were measured with Reflotron (Roche, Mannheim, Germany). Magnetic resonance imaging (MRI) was performed on a horizontal 4.