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[Establishment of method for rat hepatocyte primary culture].

AIM
To explore the culture method of rat hepatocyte between two collagen gel layers on a sandwich configuration and observe the function and morphological characteristics of the hepatocyte, which will be used in evaluation the effect of traditional Chinese Medicine on cytochrome P450.


METHODS
Rat hepatocyte were isolated by two-step in situ collagenase perfusion method; the hepatocyte were seeded in dishes coated with type I rat tail collagen, culture medium was added and changed daily after gelation. The morphological characteristics of the hepatocyte were observed and biochemical index were tested. The drug effect on the expression of CYP3A was determined by reverse transcriptase-polymerase chain reaction (RT-PCR).


RESULTS
Rat hepatocytes were successfully attached in gel. BUN and Alb excretion from cell could be tested in the culture period, however, the release of LDH content were lower than the culture system without collagen gel. The typical cellular morphological characteristics of cultured hepatocytes could be observed. PCN increased CYP3A mRNA expression in dose-dependent manner, while the expression of GAPDH wasn't affected.


CONCLUSION
Rat hepatocyte sandwich culture can maintenance the cell function and activity, which simulate the environment that more closely in vivo, especially the activity of drug metabolism enzymes.