D Li1, J Sun, H Sun, F Li, F Liu, S Liu. 1. Depatment of General Surgery, Xuanwu Hospital, Capital University of Medical Sciences, Beijing 100053.
Abstract
OBJECTIVE: To determine if the bile salt induces hepatocyte apoptosis in vitro. METHOD: Hepatocytes were isolated by in situ collagenase perfusion and plated in 6 well flat bottom with DMEM/F12, 0.5 micro/ml insulin. Two hours later, glycochenodeoxcholate (GCDC) 25, 50, 100, 200, 300 micromol was added and cells evaluated by DNA-PI staining FACS and terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) 3, 6, 9, 12, 18, 20, 24 hours later. Agarose gel electrophoresis of DNA extracted from hepatocytes after incubation with GCDC using various experimental conditions. RESULT: The hepatocytes treated with GCDC have a high apoptotic rate as compared with the controls. 100 micromol GCDC treated 24 hours, nearly 61.81% of hepatocytes were apoptotic by FACS evaluation. The TUNEL showed that apoptotic hepatocytes had less cell volume and stained with brown by Biotin-11-dUTP. DNA ladder of bile-salt treated hepatocytes were shown in agarose gel electrophoresis. CONCLUSION: The mechanism of hepatic injury during obstructive jaundice is related to bile salt caused hepatocyte apoptosis.
OBJECTIVE: To determine if the bile salt induces hepatocyte apoptosis in vitro. METHOD: Hepatocytes were isolated by in situ collagenase perfusion and plated in 6 well flat bottom with DMEM/F12, 0.5 micro/ml insulin. Two hours later, glycochenodeoxcholate (GCDC) 25, 50, 100, 200, 300 micromol was added and cells evaluated by DNA-PI staining FACS and terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) 3, 6, 9, 12, 18, 20, 24 hours later. Agarose gel electrophoresis of DNA extracted from hepatocytes after incubation with GCDC using various experimental conditions. RESULT: The hepatocytes treated with GCDC have a high apoptotic rate as compared with the controls. 100 micromol GCDC treated 24 hours, nearly 61.81% of hepatocytes were apoptotic by FACS evaluation. The TUNEL showed that apoptotic hepatocytes had less cell volume and stained with brown by Biotin-11-dUTP. DNA ladder of bile-salt treated hepatocytes were shown in agarose gel electrophoresis. CONCLUSION: The mechanism of hepatic injury during obstructive jaundice is related to bile salt caused hepatocyte apoptosis.
Authors: Fang Wang; Hong-Yi Zhou; Lan Cheng; Gang Zhao; Ji Zhou; Li-Ying Fu; Wei-Xing Yao Journal: World J Gastroenterol Date: 2003-02 Impact factor: 5.742