Effect of splenectomy on liver sinusoidal cells after 70% liver resection in a mouse model.

ABSTRACT: In this work, the effect of splenectomy (SE) on liver sinusoidal cells was studied. For this purpose in the animals were reproduced splenectomy, and after 7 days 70% liver resection (PH) was performed. Animals in which the spleen was intact and only 70% liver resection was performed served as a comparison group. Animals in which only the spleen was removed also served as controls. The animals were removed from the experiment after 24 hours, 3 and 7 days. Liver sinusoidal cells were isolated from the livers of control animals as well as from the liver after 70% resection by magnetic sorting using the CD146 beads. RNA was isolated from the obtained liver sinusoidal cells and used for transcriptome analysis. Liver sinusoidal cells in the regenerating liver were transcriptomically enriched for signaling pathways associated with monocyte migration, cell adhesion and cell death. Animals in which the spleen was intact and only 70% liver resection was performed served as a comparison group. Animals in which only the spleen was removed also served as controls. The animals were removed from the experiment after 24 hours, 3 and 7 days. Liver sinusoidal cells were isolated from the livers of control animals as well as from the liver after 70% resection by magnetic sorting using the CD146 beads. RNA was isolated from the obtained liver sinusoidal cells and used for transcriptome analysis. Liver sinusoidal cells in the regenerating liver were transcriptomically enriched for signaling pathways associated with monocyte migration, cell adhesion and cell death. The mouse liver was dissected, minced and incubated in 0.1% solution of type I and IV collagenases (PanEco, Russia) at 37° C and horizontal shaking for 30 minutes. The suspension was passed through a 100 µm nylon strainer (SPL Life Sciences, Korea) and washed twice from the enzymes (300 g at 20 °С for 10 minutes). The cells were resuspended in 30 mL of phosphate-buffered saline and centrifuged at 50 g for 3 minutes. As a result, parenchymal cells of the liver (hepatocytes) were sedimented and the non-parenchymal cell types (including liver macrophages, the Kupffer cells) remained in the supernatant. To enrich the stromal cell fraction with macrophages, the supernatant was mixed with Lympholyte®-M density separation medium (Cedarlane, Canada) and centrifuged at 400g and 20 °C for 30 minutes. The obtained fraction predominantly consisted of Kupffer cells. Cell number and viability were assessed in a TC20 analyzer (Bio-Rad). The suspension underwent immunomagnetic sorting on a manual MidiMACS™ Separator using LS Columns (Miltenyi Biotec, Germany) with Anti-CD146 MicroBeadsUltraPure magnetic microparticles (Miltenyi Biotec) in accordance with the manufacturer’s recommendations. Total RNA was isolated from endothelial cells obtained by magnetic sorting for CD146 using the RNeasy Plus Mini Kit (QIAGEN, Germany). Transcriptome analysis was performed using microarray Clariom™ S Assay, mouse (Applied Biosystems™). Next, reverse transcription using the WT Amplification Kit was performed to synthesize first-, second-strand cDNA, cRNA and ss-cDNA incubating at appropriate temperatures and purifying via magnetic beads. Then, ss-cDNA was fragmented, labeled with biotin, hybridized to the array at 45°C for 16 hours, followed by washing. GeneChips were scanned using the Affimetrix GeneChip Scanner 3000 7g. The data were analyzed with Transcriptome Analysis Console (TAC) Software version 4.0.2 using Affymetrix default analysis settings and RMA algorithm

ORGANISM(S): Mus musculus (mouse)

SUBMITTER:  

PROVIDER: S-BSST1922 | biostudies-other |

REPOSITORIES: biostudies-other