[Differentiation of hepatic oval cell into mature hepatocyte induced by hepatic stellate cells].
OBJECTIVE
To investigate the role of hepatic stellate cells in the differentiation of hepatic oval cells into adult hepatocyte.


METHODS
The oval cell were cocultured with primary hepatic stellate cells (HSC) in the same well (M-coculture) or separately cultured with HSC by millIcell (S-coculture). Oval cells were cultured alone as control; the expression of adult hepatocyte marker HNF-4alpha, albumin, and oval cell marker AFP, CK-19 in each group were detected by real-time PCR and western-blot. Phenotype changes were observed by transmission electron microscope (TEM); PAS staining was used to detect the quantity of glycogen granule in oval cell. Albumin level in supernatant was detected using ELISA kit.


RESULTS
(1) The relative level of HNF-4alpha and albumin mRNA expression compared with pre-coculture: M-coculture: HNF-4a: 1.9+/-0.2, 10.7+/-1.2, 12.0+/-1.3; albumin: 5.7+/-1.6, 110.7+/-13.7, 173.6+/-22.3. S-coculture: 1.4+/-0.1, 3.2+/-0.6, 8.9+/-1.4 times; albumin: 2.9+/-1.4, 22.3+/-8.5, 96.3+/-16.3. The relative level of HNF-4a and albumin mRNA expression in coculture group (M- and S-coculture) were higher than control group (LSD-t: 32.98, 10.08, 13.38, 7.96; P less than 0.01); and a higher level of HNF-4a and albumin was found in M-coculture group compared to S-coculture group (LSD-t: 32.98, 25.65; P less than 0.01). The relative level of AFP and CK-19 mRNA expression compared with pre-coculture: M-coculture: 1.1+/-0.2, 0.2+/-0.0, 0.0+/-0.0; S-coculture group: AFP: 1.0+/-0.2, 0.2+/-0.1, 0.1+/-0.0; CK-19: 0.6+/-0.1, 0.1+/-0.0, 0.0+/-0.0; control group: AFP: 1.0+/-0.1, 1.0+/-0.1, 1.1+/-0.1, CK-19: 1.0+/-0.1, 1.1+/-0.1, 1.0+/-0.1. The relative level of AFP and CK-19 mRNA expression in coculture group (M- and S-coculture) were lower than that in control group (LSD-t: 37.99, 34.50, 13.59, 22.46; P less than 0.01). (2) The albumin secretion was detected in M-coculture: 14 day: (15.30+/-0.09) ng/ml, 21: (20.98+/-0.12) ng/ml; S-coculture: 14 day: (11.41+/-0.13) ng/ml, 21 day:(15.12+/-0.17) ng/ml. (3) It showed more organelles such as endoplasmic reticulum, mitochondrion and Golgi apparatus in oval cells cocultured with HSC. And cholangiole-like structure appeared between oval cells cocultured with HSC. (4) PAS staining showed glycogen granules could be observed in coculture groups.


CONCLUSIONS
HSC can induce differentiation of oval cell into mature hepatocyte.
Chen
Lin
L
Department of Hepatic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Chen
Xiao-Ping
XP
Zhang
Wei
W
Liang
Hui-Fang
HF
Lin
You-Zhi
YZ
Dong
Han-Hua
HH
Zhou
Qiao-Dan
QD
D000418:Albumins / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D018920:Coculture Techniques; D055166:Hepatic Stellate Cells; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D008099:Liver / Q000166:cytology*; D008297:Male; D018613:Microscopy, Confocal; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D000509:alpha-Fetoproteins / Q000096:biosynthesis / Q000235:genetics
D000418:Albumins; D012333:RNA, Messenger; D000509:alpha-Fetoproteins