[Establishment and growth regulation of rat hepatocyte colony in vitro].
OBJECTIVE
To investigate the conditions essential for culturing hepatocytes colony in vitro, and to study the growth regulation mechanisms of hepatocyte colony.


METHODS
Rat hepatocytes were isolated by two-step in situ preperfusion and collagenase circulatory perfusion. The effects of hepatopoietin (HPN), nicotinamide (NA) and dimethyl sulfoxide (DMSO) on DNA synthesis, mitotic activity, morphology (under inverted microscope) and utrastructure (under TEM) of the hepatocytes were investigated in chemically defined culture medium.


RESULTS
The time course of DNA synthesis in cultured hepatocytes showed that 3H-TdR incorporation was dramatically enhanced by 10 mmol/L NA, presenting 2 peaks at 60 and 84 h respectively. The plateau of DNA synthesis was diminished in the presence of DMSO, but a peak occurred again at 132 h upon NA treatment. After cell culture in the presence of HPN, NA, and DMSO for 72 h, the hepatocytes presented sustained regular bipolar mitosis, with considerable mitotic activity at 168 h. The growth characteristics of hepatocyte colony, in addition to its potential for expansion, were captured by both light and electron microscopy on day 28 of cell culture.


CONCLUSIONS
The reciprocal actions of NA and DMSO can control the proliferation of HPN-stimulated hepatocytes, which can be used for studying human hepatocyte metabolism, cytotoxicity, biotransformation and mutagenesis, and may provide experimental evidences for the treatment of liver failure and genetic liver diseases with in vitro hepatocyte clones.
Tang
Xi-feng
XF
Department of Laboratory Medicine, Nanfang Hospital, First Military Medical University, Guangzhou 510515, China. tangxf@fimmu.com
Zhou
Dong-yao
DY
Xu
Zhi-min
ZM
Zhou
Xiao-dong
XD
D000818:Animals; D002455:Cell Division / Q000502:physiology; D004247:DNA / Q000096:biosynthesis; D017228:Hepatocyte Growth Factor / Q000502:physiology*; D022781:Hepatocytes / Q000166:cytology / Q000502:physiology* / Q000648:ultrastructure; D006801:Humans; D051381:Rats
D017228:Hepatocyte Growth Factor; D004247:DNA