OBJECTIVETo establish a method for simultaneous isolation and primary culture of rat hepatocytes, hepatic stellate cells, Kupffer's cells and hepatic sinus endothelial cells.

METHODSBy combining in situ perfusion, in vitro perfusion, density gradient centrifugation and differential adhesion, primary rat hepatocytes, hepatic stellate cells, Kupffer's cells and hepatic sinus endothelial cells were obtained. The purity of these cells were assessed with morphological observation, immunofluorescent staining and ink phagocytosis assay.

RESULTSWe successfully obtained the 4 primary cells simultaneously by combining in situ perfusion with in vitro perfusion, density gradient centrifugation, and differential attachment. The cell yield rate, cell viability and purity all met requirements for the subsequent cell experiment.

CONCLUSIONThe combined cell isolation and culture method is feasible to isolate primary rat hepatocytes, hepatic stellate cells, Kupffer's cells and hepatic sinus endothelial cells simultaneously.

Animals ; Cell Culture Techniques ; Cell Separation ; Endothelial Cells ; cytology ; Hepatic Stellate Cells ; cytology ; Hepatocytes ; cytology ; Kupffer Cells ; cytology ; Male ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the inhibitory effects of spironolactone against hepatic sinusoid angiogenesis in rats with hepatic fibrosis.

METHODSTwenty-four male Wistar rats were randomly divided into sham-operated group, bile duct ligation (BDL) group, and BDL+SP group in which the rats received daily spironolactone injection (20 mg/kg) the day after BDL. Four weeks after the operation, the rats were sacrificed for examination of liver histology using Masson staining and the expression of vascular endothelial growth factor A (VEGF-A) mRNA in the liver using real-time quantitative PCR. Immunohistochemistry was used to detect the expression of von Willebrand factor (vWF) in the hepatic tissues.

RESULTSSpironolactone significantly inhibited liver fibrogenesis in rats after BDL (METAVIR liver fibrosis scores 2.84∓0.44 vs 19.73∓3.54, P=0.00). Real-time PCR and immunohistochemistry showed that compared with BDL group, spironolactone treatment significantly inhibited the expression of VEGF-A mRNA (0.71∓0.12 vs 1.75∓0.15, P=0.00) and vWF (1.15∓0.09 vs 3.08∓0.17, P=0.00) in the liver. The expression of VEGF-A mRNA was highly correlated with the expression of vWF (r=0.890, P=0.000).

CONCLUSIONSpironolactone can inhibit hepatic sinusoid angiogenesis in rats with BDL-induced hepatic fibrosis by inhibiting the expression of VEGF-A.

Animals ; Hepatic Veins ; pathology ; Liver Cirrhosis, Experimental ; metabolism ; pathology ; Male ; Neovascularization, Pathologic ; drug therapy ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar ; Spironolactone ; pharmacology ; Vascular Endothelial Growth Factor A ; metabolism