초록 ▼

1. Rat 과 mouse를 이용한 간세포의 일차배양 인체에서 획득 가능한 조직의 성상을 모델로 하여, perfusion을 이용 하지않는 세포의 분리 및 배양 방법을 개발2. 인간 유래 간세포의 배양 실험동물을 이용하여 확립한 세포의 분리 및 배양 방법을 응용하여, 인간유래 간세포의 배양 기술 개발3. 분석 배양 간세포의 약리학적 세포생물학적 분석 배양 간세포의 형태학적 분석, 약물 대사능력 및 호르몬 반응성을 검토하기위해, TCDD에 의해 cytochrome P-450 의 induction 및 AIB uptake, prote

1. Rat 과 mouse를 이용한 간세포의 일차배양 인체에서 획득 가능한 조직의 성상을 모델로 하여, perfusion을 이용 하지않는 세포의 분리 및 배양 방법을 개발2. 인간 유래 간세포의 배양 실험동물을 이용하여 확립한 세포의 분리 및 배양 방법을 응용하여, 인간유래 간세포의 배양 기술 개발3. 분석 배양 간세포의 약리학적 세포생물학적 분석 배양 간세포의 형태학적 분석, 약물 대사능력 및 호르몬 반응성을 검토하기위해, TCDD에 의해 cytochrome P-450 의 induction 및 AIB uptake, protein assay, TAT activity test, albumin합성, DNA 합성등 기능학적 분석을 실시하였음.

Abstract ▼

Hepatocyte has many important functions to maintain whole body homeostasis. For the studies on liver, many in vitro models were propose. These included isolated liver perfusion, liver slice, cell line culture, isolated hepatocyte and primary hepatocyte culture. Compared with other methods, primary h

Hepatocyte has many important functions to maintain whole body homeostasis. For the studies on liver, many in vitro models were propose. These included isolated liver perfusion, liver slice, cell line culture, isolated hepatocyte and primary hepatocyte culture. Compared with other methods, primary hepatocyte culture has many merits. It can be cultured for several weeks to support long term studies and has many liver specific functions which are useful for pharmacological and toxicological studies. The current methods for hepatocyte isolation are based collagenase perfusion techniques. But this method are useful only for whole organ and large organ mass which has an comparable intact blood vessel circuit. So, to isolate human hepatocyte, a large mass (minimum 25g) was required. Because the organ donation is not prevail in our country, it is very difficult to acquire large sample of human liver. Based on this problem, we tried to isolate hepatocytes using small size sample (1∼2g). Many factors which were influencing on hepatocyte isolation procedure were examined and a standard procedure was acquired for rat hepatocyte. Isolated hepatocytes were applied to culture and proven to have intact morphological characteristics. In the case of human, a standard procedures for rat hepatocytes was slightly modified to consider the conditions of human sample. The morphological integrities were also proven in both isolated and cultured hepatocytes. During cultivation, their functional integrities including drug metabolizing capacities, hormonal responsibilities, albumin synthesis, DNA synthesis, cell mass maintenance have been proven. The usefulness of primary human hepatocyte culture are really enormous in the studies of medical, pharmacological, toxicological and biological field. The method and systems established in present paper will contribute and support these scientific studies.

목차 Contents

• 제1장 서 론...21
• 제2장 재료 및 방법...25
• 1) 재료...25
• 2) Rat 간세포의 분리...25
• 3) 인체 유래 간세포의 분리...26
• 4) 세포 계수 및 생존률...27
• 5) 세포 배양의 조건...27
• 6) 분리 세포와 배양 세포의 현미경적 관찰...28
• 7) 전자현미경적 관찰...28
• 8) 단백질 정량...29
• 9) TCDD induction 및 EROD actibity 측정...29
• 10) AIB uptake determmation...29
• 11) TAT activity determmation...30
• 12) ELISA에 의한 albumin 정량...30
• 13) DNA 합성량 측정...31
• 14) 통계처리...32
• 제3장 결 과...33
• 1) Rat liver 에서의 간세포 isolation...33
• 2) Rat hepatocyte의 primary culture...37
• 3) Human liver sample에서의 간세포 isolation...44
• 4) Human hepatocyte의 primary culture...53
• 5) Isolated rat hepatocyte의 전자현미경 관찰...53
• 6) Phalloidin에 의한 protrusion 형성...54
• 7) Rat hepatocyte culture시 protein양 변화...54
• 8) Dexamethasone에 의한 TAT의 induction...54
• 9) Dexamethasone과 glucagon에 의한 AIB uptake의 induction...56
• 10) TCDD에 의한 EROD induction...58
• 11) Human hepatocyte에서의 albumin의 합성...62
• 12) Hepatocyte culture에서의 DNA 합성...63
• 제4장 고 찰...67
• 참 고 문 헌...71
• 부 록 실험에 사용된 salt solution 과 media...77