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Establishment of the isolated rat hepatocytes culture system to study drug metabolizing enzyme

Title
Establishment of the isolated rat hepatocytes culture system to study drug metabolizing enzyme
Authors
조선남
Issue Date
1997
Department/Major
대학원 약학과
Keywords
Establishmenthepatocytes culturedrug약학
Publisher
이화여자대학교 대학원
Degree
Master
Abstract
Cytochrome P450 1A1 has been studied with cell lines and perfused rat liver. Because of the expected usefulness of isolated rat hepatocytes in the drug metabolism study, it was necessary to study the expression of cytochrome P450 1A1 in the isolated hepatocytes. Hepatocytes from adult male Sprague-Dawley rats were isolated by the two-step collagenase perfusion procedure. The effect of TCDD, retinoic acid, dexamethasone, and estrogen on cytochrome P450 1A1 expression had been studied through message by each mRNA. Cytochrome P450 1A1 expression had been reported to be decreased, when the isolated hepatocytes were cultured. But the level of mRNA was detected by RTPCR and the level was constantly high without inducer. After cells were cultured in phenol red free MEM, the level of cytochrome P45O 1A1 mRNA was decreased slowly. Phenol red free MEM was used because estrogenic effect of phenol red had been suspected to induce the cytochrome P450 1A1 expression. After 1 month culture, cells were treated with chemicals, harvested and the level of cytochrome P45O 1A1 mRNA was analyzed by RTPCR. The level of cytochrome P45O 1A1 mRNA was increased by TCDD treatment. When retinoic acid or dexamethasone was treated alone, the level of cytochrome P45O 1A1 mRNA was increased. When retinoic acid was treated with TCDD concomitantly, retinoic acid inhibited the induction by TCDD. Estrogen (estradiol) was inductive alone, and it's effect was inhibited by tamoxifen treatment. When cells were treated with TCDD 2 days after colture, ethoxyresorufin-0-deethylase activity was increased compared to that of control. In order to see c-fos gene expression of the cell, the level of c-fos mRNA was measured by RTPCR following the fetal bovine serum treatment. c-fos expression was increased for 2 hours with fetal bovine serum treatment. The elevated level of c-fos mRNA with fetal bovine serum treatment was maintained for a while when cells were deprived of fetal bovine serum, then decreased. DMSO effect on cfos was examined in the presence or absence of fetal bovine serum. c-fos expression was increased with DMSO treatment, and it's effect was greater in the presence of fetal bovine serum than in the absence of fetal bovine serum. The elevated level of c-fos mRNA was decreased within 24 hours.;Cytochrome P450 lAl은 cell line과 관류한 백서의 간에서 많은 연구가 이루어져 왔으며, 분리된 간세포가 약물대사 연구에 유용하게 쓰일 수 있을 것으로 사료되어, 분리된 간세포에서 cytochrome P450 lAl 유전자의 발현조절에 관한 연구가 필요하게 되었다. 백서로부터 2단계 collagenase 관류법에 의하여 간세포를 분리하여 배양하였으며, TCDD, retinoic acid, dexamethasone, estradiol에 의한 cytochrome P450 lAl 유전자의 발현조절을 cytochrome P450 lAl mRNA의 양을 RTPCR의 방법으로 측정하여 관찰하였다. 분리된 간세포를 배양하게되면 발현을 유도하는 약물을 투여하더라도 cytochrome P450 lAl 유전자 발현이 나타나지 않는다고 보고되어 왔으나, 약물 투여 없이도 cytochrome P45O lAl mRNA 의 양이 높게 나타났다. 배지를 phenol red 없는 MEM으로 바꾸어 주었을 때, cytochrome P45O lAl mRNA의 양은 서서히 감소하였다. Phenol red의 estrogen과 같은 효과는 cytochrome P450 lAl 유전자 발현을 유도할 수도 있기 때문에 phenol red 없는 MEM을 계속해서 사용 하였다. 1 개월 이상 배양한 후에, 약물을 처치하고 cytochrome P45O lAl mRNA의 양을 RTPCR에의한 방법으로 측정하였다. Cytochrome P45O lAl mRNA의 양은 TCDD를 투여하였을 때 증가하였다. Retinoic acid를 단독으로 투여하였을 때 cytochrome P45O lAl mRNA의 양은 증가하고, retinoic acid를 TCDD와 병용투여 하였을 때는 TCDD가 cytochrome P450 lAl의 유전자 발현을 유도하는 것을 감소시켰다. Dexamethasone을 단독으로 투여 하였을 때, cytochrome P45O lAl mRNA의 양이 증가하였다. Estradiol은 단독으로 투여하였을 때 cytochrome P45O lAl mRNA의 양을 증가 시켰으나, tamoxifen과 병용 투여하였을 때 cytochrome P45O lAl mRNA의 양이 감소하였다. 간세포를 분리한 직후부터, ethoxyresorufin-0-deethylase (EROD) 활성을 측정하여 배양하는 동안 EROD활성을 측정하였을 때, 간세포를 분리한 직후에는 EROD활성이 높았으나, 배양을 계속하는 동안 급격히 감소하고, 3일이 지났을 때는 활성이 나타나지 않았다. 배양시작후 48시간이 지난후에, TCDD로 20 시간 처치한 세포에서는 EROD 활성이 비교군에 비해서 증가하였다. 간세포의 early gene의 발현을 연구하기 위하여, c-fos 유견자의 발현을 fetal bovine serum (FBS)을 투여한 추에 c-fos mRNA의 양을 RTPCR의 방법으로 측정하였다. c-fos 유전자 발현은 FBS투여로 인해 2시간 까지 증가하였으며, FBS를 제거하였을 때에도 증가된 상태를 유지하다가 그 이후에는 감소하였다. FBS가 있을 때와 없을 때 DMSO를 투여했을 때에는 FBS가 있을 때 c-fos 유전자 발현의 증가가 많았다.
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