There is a growing interest in the application of primary hepatocytes for treatment of liver diseases in humans and for drug development. Several studies have focused on long-term survival and di-differentiation blocking of primary hepatocytes in an in vitro culture system. Therefore, the present study also aimed to optimize an in vitro culture system using primary rat hepatocytes. Primary rat hepatocytes from 6-week-old male Crl:CD rats were isolated using a modified two-step collagenase perfusion. Healthy 3.5 × 106 primary rat hepatocytes were seeded into a 2 dimensional (2D) culture in a 25T culture flask coated with collagen type I or into a 3D culture in a 125-ml spinner flask for 7 days. Production of plasma protein (ALB and TF), apoptosis (BAX and BCL2), and CYP (CYP3A1) related genes were compared between the 2D and 3D culture systems. The 3D culture system had an advantage over the 2D system because of the relatively high expression of ALB and low expression of BAX in the 3D system. However, the level of CYP3A1 did not improve in the 3D culture with and without the presence of a dexamethasone inducer. Therefore, 3D culture has an advantage for albumin production and primary rat hepatocyte survivability, but a low expression of CYP3A1 indicated that primary rat hepatocytes require a high–density culture for stress reduction by continuous flow.

본 연구의 목적은 양파추출물의 간보호 및 항산화 효과를 조사하기 위함이다. 간 손상을 유발시키는 t-BHP (1.5 mM) 존재하에 간세포를 0, 0.01, 0.05, 0.1 및 0.3 mg/ml의 다양한 농도의 양파추출물로 1시간 동안 일차배양하였다. t-BHP는 GOT와 LDH 활성 및 TBARS 농도를 증가시켰으며 MTT값은 감소시켰다. 0.05 mg/ml 농도의 양파추출물 첨가는 t-BHP에 의해 증가된 GOT 및 LDH 활성을 감소시켰으며 0.1 mg/ml 농도의 양파추출물은 t-BHP에 의해 감소된 MTT 값을 증가시켰다. 또한 0.01 mg/ml 농도의 양파추출물 첨가는 t-BHP에 의해 증가된 TBARS 농도를 감소시켜 양파추출물이 t-BHP에 의해 유발된 간손상과 지질과산화를 억제시켰다. t-BHP 처리는 간세포의 catalase, GSH-Px 및 GSH-Rd 활성을 현저히 감소시켰다. 그러나 0.1 mg/ml 농도의 양파추출물 첨가는 t-BHP에 의해 감소된 catalase GSH-Px 및 GSH-Rd 활성을 증가시켰으며 특히 catalase 활성은 t-BHP 무첨가군 수준까지 증가시켰다. 또한 hydroxyl radical을 생성하는 Fenton 시약의 존재하에 plasmid DNA를 양파추출물과 함께 배양한 결과 양파추출물은 농도 의존적으로 hydroxyl radical에 의해 유도된 single-strand 절단을 억제하였다. 이상과 같이 간세포 일차배양에서 양파추출물은 t-BHP에 의해 유발된 간독성, 간세포 생존율 감소, 지질과산화를 농도 의존적으로 억제시켰고 또한 t-BHP에 의해 억제된 GSH-Px, GSH-Rd 및 catalase의 활성을 증가시켰다. 이와 같이 양파추출물의 간보호 및 항산화 효과는 항산화 효소 특히 catalase의 활성 증가와 hydroxyl radical에 의해 유도된 산화억제 및 이에 따른 지질과산화 억제에 기인하는 것으로 사료된다.

The objectives of present study were to investigate the effects of benzo[a]pyrene(BaP) on cytotoxicity, lipid peroxidation and antioxidant enzymes in rat hepatocyte primary culture. Primary cultures of rat hepatocytes were incubated for 24 hr, 48 hr or 72 hr in the presence of various concentrations (0, 10, 20, 30, 50 or 100 μM) of BaP. Cytotoxicity and cell viability were determined by measuring glutamic oxaloacetic transaminase(GOT) activity, lactate dehydrogenase(LDH) activity and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) value. Lipid peroxidation was evaluated using thiobarbituric acid reactive substances(TBARS) assay. Effects on antioxidant system were determined by measuring glutathione peroxidase(GPx) activity, glutathione reductase(GR) activity and glutathione concentration. Activities of GOT and LDH, MTT value as well as TBARS concentration were not affected by up to 100 μM of BaP for 24 hr incubation. However, BaP at the concentration of 50 μM for 48 hr incubation or at the concentration of 30 μM for 72 hr incubation began to increase LDH activity and TBARS concentration but decrease MTT value, representing that BaP caused cytotoxicity and decreased cell viability in dose- and time-dependent manners. GPx activity began to be decreased by BaP at the concentration of 50 μM for 72 hr incubation. Whereas, GR activity began to be decreased by BaP at the concentration of 20 μM for 72 hr incubation. Glutathione concentration began to be decreased by BaP at the concentration of 20 μM for 72 hr incubation and was further reduced to 90% by 100 μM of BaP. These results demonstrate that BaP caused cytoctoxicity and decreased cell viability by increasing lipid peroxidation and decreasing glutathione concentration as well as activities of GPx and GR.