Functional evaluation of bioartificial liver using RT-PCR.
In order to evaluate functions of a bioartificial liver (BAL), containing porcine hepatocytes in a hollow fiber cartridge, some chemical loading tests have been employed. However the kinds of functions that can be evaluated by chemical loading tests are limited. We desire versatile methods to estimate various BAL functions. The purpose of this report is to propose a method using the reverse transcription polymerase chain reaction (RT-PCR) for functional evaluation of a BAL. In vitro perfusion culture of a BAL cartridge was carried out using 20% of human whole blood. At pre-determined periods of perfusion culture, hepatocytes were taken from the cartridge and mRNA was extracted from the hepatocytes. The mRNA expression levels of albumin and cytochrome P450 (CYP3A29) were determined by RT-PCR method. In order to quasi-quantitatively determine the time courses of mRNA expression levels during the perfusion culture, PCR of target DNA was carried out by co-amplification with its competitor DNA that was constructed by means of the partial deletion of target DNA. The results showed that the amounts of both albumin and cytochrome P450 mRNA rapidly decreased during the initial few days' perfusion culture and remained at a constant level for the following week. This fact corresponded with the analysis of lidocaine metabolic functions via pharmacokinetics in our previous study. Additionally, the RT-PCR is so sensitive that we can measure much more minute quantity of various proteins. Consequently, this method is useful for comprehensive evaluation of BAL functions.
Fujita
Satoshi
S
Institute for Frontier Medical Sciences, Kyoto University, Japan.
Ueda
Yuichiro
Y
Ko
In Kap
IK
Paek
Hyun Joon
HJ
Sajiki
Toshinobu
T
Ikai
Iwao
I
Yamaoka
Yoshio
Y
Ikada
Yoshito
Y
Iwata
Hiroo
H
D000418:Albumins / Q000737:chemistry; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry; D001667:Binding, Competitive; D001698:Biomedical Engineering / Q000379:methods*; D051544:Cytochrome P-450 CYP3A; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D015723:Gene Library; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000473:pathology; D017114:Liver Failure, Acute / Q000628:therapy; D019164:Liver, Artificial; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013552:Swine; D013997:Time Factors; D023822:Tissue Engineering
D000418:Albumins; D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D004247:DNA; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating