• Archives of Pharmacal Research
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• 제21권6호
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• pp.718-722
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• 1998

Scopoletin (7-hydroxy-6-methoxycoumarin), a coumarin, was isolated from the aerial part of Solanum lyratum Thunb. by the activity-guided fractionation employing carbon te trachloride-intoxicated primary cultured rat hepatocytes as a screening system. Its hepatoprotective activity was first evaluated by measuring the release of glutamic pyruvic transaminase and sorbitol dehydrogenase from carbon tetrachloride-intoxicated rat hepatocytes into the culture medium. Scopoletin significantly reduced the releases of glutamic pyruvic transaminase and sorbitol dehydrogenase from the carbon tetrachloride-intoxicated primary cultured rat hepatocytes by 53% and 58%, respectively, from the toxicity in a dose-dependent manner over concentration ranges of 1mcM to 50mcM. Further studies revealed that at the concentration of 10mcM, scopoletin significantly preserved glutathione content by 50% and the activity of superoxide dismutase by 36% and also inhibited the production of malondialdehyde to the degree as seen in the control.

• 한국미생물·생명공학회지
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• 제28권4호
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• pp.209-213
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• 2000

The cultured mycelia of Ganoderma lucidum were extracted and the extract was separated into six fractions by organic solvent fractionation. The antihepatotoxic activity of all the fractions was evaluated by measuring activities of glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT). Among the fractions tested, the high-polarity fractions such as aqueous and n-butanol fractions significantly reduced activities of GPT and GOT in CCl4- and galactosamine-intoxicated rat primary hepatocytes. When intracellular synthetic activities were measured by pulsing the rate primary cultured hepatocytes with [3H]-uridine and [3H]-leucine, activities of DNA, RNA and protein. When direct toxicities of the fractions were measured against human hepatoma(SK-Hep-1), the non-polarity fractions such as n-butanol and ethyl acetate fractions showed potent direct cytotoxicities even at the concentration of 1 $\mu\textrm{g}$/ml. These data showed that Ganoderma lucidum has hepatoprotective and hepatotoxic recovery principles in its mycelia.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.305-306
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• 2003

To treat fulminant hepatic failure (FHF) patients, various extracorporeal bioartificial liver (BAL) systems have been developed. Several requirements should be met for the development of BAL systems: hepatocytes should be cultured in a sufficiently high density; their metabolic functions should be of a sufficiently high level and duration; and the BAL systems module should permit scaling-up and aseptic handling. Several investigators have found that freshly isolated primary hepatocytes can be cultured into three dimensional, tightly packed, freely suspended, multicellular aggregates, or spheroids. These specialized cell structures exhibited enhanced liver specific functions and a prolonged differentiated state compared to cells maintained in a monolayer culture. Cells in spheroids appear to mimic the morphology and ultrastructure of the in vivo liver lobule. The ability of hepatocytes to organize into three-dimensional structures was hypothesized to contribute to their enhanced liver-specific activities. In this study, the ammonia removal rate and urea secretion rate of pig hepatocytes spheroids encapsulated in Ca-alginate bead were determined. A packed-bed bioreactor with encapsulated pig hepatocytes was devised as BAL support system. The efficacy of the system was evaluated in vitro.

• Biotechnology and Bioprocess Engineering:BBE
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• 제3권2호
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• pp.96-102
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• 1998

Primary hepatocytes of small animals such as rat and rabbit were often used for the study of extracorporeal liver support systems. Freshly isolated rat hepatocytes form spheroids within tow days when cultivated as suspension in spinner vessels. These spheroids showed enhanced liver specific functions and more differentiated morphology compared to hepatocytes cultured as monolayers However, shear stress caused by continuous agitation deteriorated spheroids gradually. In this work we immobilized spheroids to prolong liver specific activities. First, hepatocyte spheroids were suspended in collagen solution containing calcium chloride and then dropped into alginate solution. A thin layer of calcium alginate was formed around the droplet and then was removed after the inner collagen was gelled by treatment of sodium citrate buffer. Spheroids embedded in collagen-gel bead maintained liver specific functions such as albumin secretion rate longer than hepatocyte spheroids exposed to shear stress. Therefore, we suggest that this immobilization technique may offer an effective long-term hepatocyte cultivation and facilitase the development of a bioartificial liver support device.

• 한국가축번식학회지
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• 제20권3호
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• pp.259-269
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• 1996

To establish whether or not androgens is responsible for the induction of vitellogenin(Vg) synthesis and secretion, primary hepatocytes prepared from immature eels were used. The results are follows: 1. Eel hepatocytes were prepared using a collagenase perfusion technique. The isolated cells attached efficiently to fibronectin-coated dishes and subsequently formed monolayers in serum-free medium. These cultures maintained in medium for 10 days with minimal cell loss. 2. Estradiol-17$\beta$(E2) alone was insufficient to induce Vg synthesis. The combination of E2 with methyltestosterone(MT) markedly stimulated Vg synthesis. High vg production occurred in MT concentration from 10-6~10-5M in the presence of E2 (10-6M). Testosterone and androsterone were also effective, but progesterone was not effective in inducing Vg synthesis. Neither MT alone nor testosterone and androsterone alone had any effect on Vg synthesis. 3. E2-primed hepatocytes showed Vg synthesis in both media with and without hormones 1 day after culture. In the cultures with the vehicle, MT, or progesterone, the rate of synthesis seemed to decrease with time. But the combination of E2 and MT showed an intense increase in Vg synthesis. Hepatocytes isolated from E2-primed eels also required androgens for continuating of Vg synthesis. 4. These results demonstrate that androgens act together with E2 in synthesis and secretion of eel Vg.

• Fisheries and Aquatic Sciences
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• 제1권1호
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• pp.19-23
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• 1998

Effects of pituitary and thyroid hormones on estradiol-induced vitellogenin (VTG) induction were electrophoretically examined in primary hepatocyte cultures of rainbow trout. Hepatocytes were precultured for 2 days and then estradiol-17 $\beta$ $(E_2,\;2 \times 10^{-6}M)$>, triiodothyronine $(T_3,\;10^{-8}-10^{-6}M)$, bovine growth hormone (bGH, 10-100 ng/ml), ovine prolactin (oPRL, 100-500 ng/ml), and pituitary extract (PE) of rainbow trout (0.75PE/dish) were added to the incubation medium. The hepatocytes were cultured for 7 more days. The addition of oPRL to the incubation medium was not effective in increasing VTG production at any concentrations. The addition of PE to the incubation medium with $E_2$ was not effective in increasing VTG production. The addition of bGH to the incubation medium with $E_2$ was not effective in increasing the rate of VTG production at concentrations of 10-50 ng/ml. However, a higher concentration of bGH, 100 ng/ml, increased VTG production. The various concentrations of $T_3$ were ineffective in stimulating VTG production. These results suggest that GH could be one of stimulus factors for VTG production in rainbow trout.

• 한국양식학회지
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• 제11권2호
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• pp.241-248
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• 1998

Vitellogenin(VTG) 합성에 미치는 A23187의 영향을 무지개송어의 배양 간세포를 이용하여 실험을 행하였다. 간세포는 2일간 배양한 후, Estradiol-$17^{\beta}$($E_2$, $2{\times}10^{-6}$M) 및 A23187 ($10^{-7)$~$10^{-5}$M)을 첨가하여 7일간 배양하였다. 그리고, $E_2$에 의한 VTG 합성시의 A23187이 미치는 영향에 대해서도 조사하였다. A23187이 미치는 영향에 대해서도 조사하였다. A23187 ($10^{-7)$~$10^{-5}$M)의 첨가에 의해 간세포에서의 VTG 합성은 농도의 증가에 따라 감소하였다.$E_2$에 의해 합성된 VTG는 A 23187 ($10^{-5}$M)의 첨가에 의해 대조군($E_2$만의 첨가)의 약 18%로 유의하게 감소하였다. 그러나,$E_2$제거로는 대조군의 약 47% 밖에 감소되지 않았다. 이러한 결과로 보아, 세포내의 저장 Ca은 번역 단계 또는 번역 후 단계를 조절함으로써, VTG 합성을 조절하는 것으로 추정된다.

• Archives of Pharmacal Research
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• 제13권4호
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• pp.355-358
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• 1990

In order to investigate the cellular mechanisms of hypoglycemic of brazilin, hepatocyte monolayer culture was introduced and, glycogen synthesis rate and insulin binding were measured as parameters. Glycogen synthesis and insulin sensitivity were remarkably augmented by the treatment of brazilin. Brazilin slightely increased insulin binding. Scatchard analysis revealed that this increase in insulin binding was not due to increase in the binding capacity but in binding affinity. These results suggest that the augmentation of hepatic glycogenesis and insulin sensitivity by brazilin may play an important role in the improvement of hyperglycemia.

• 한국어류학회지
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• 제12권3호
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• pp.180-185
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• 2000

플라스틱의 원재료로 이용되는 bisphenol-A (BPA)가 vitellogenin (VTG)의 합성에 미치는 영향을 넙치의 간세포를 초대 배양하여 조사하였다. 간세포는 2일간 사전 배양을 하였으며, estradiol-$17\beta(E_2)$ 및 BPA를 배양액에 동시 첨가하였다. 간세포는 6일간 배양한 후 배양액을 회수하여 SDS-PAGE로 분석하였다. BPA는 농도 의존적으로 총 단백질에 대한 VTG 비율은 증가하였으며, $10^{-6}M$과 $10^{-5}M$의 농도에서 유의한 차이를 나타내었다(P<0.05). 특히, BPA $10^{-5}M$의 농도에서의 총 단백질에 대한 VTG 비율은 26.36%로 $E_2$만을 첨가한 대조군과 유의한 차이를 나타내지 않았다. $E_2$와 BPA를 단독 또는 동시에 첨가하여 유도된 VTG합성은 배양액에 tamoxifen$(10^{-6}M)$을 첨가함으로서 $E_2$를 첨가한 대조군의 약 80%로 유의하게 억제되었다. 그리고, in vivo에서 $E_2$에 의해 유도된 VTG합성은 in vitro에서 간세포를 6일간 배양함에 따라 $E_2$를 첨가한 대조군의 약 22%까지 억제되었다. 이러한 감소효과는 BPA의 농도 의존적으로 지연되는 결과를 나타내었다. 따라서 넙치에서의 BPA는 $E_2$ receptor와의 반응을 통한 에스트로겐 작용에 의해 VTG합성을 유도하고, 난황형성기에서의 VTG 합성을 장시간 지속시킬 가능성이 있는 것으로 추측된다.

• 한국식품과학회지
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• 제35권1호
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• pp.138-143
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• 2003

쑥갓의 total methanol 추출물이 galactosamine으로 독성을 유발시킨 흰쥐의 일차배양 간세포에서 유리되는 GPT의 활성을 유의성 있게 감소시켜 간세포 보호활성을 보임을 알 수 있었다. Total methanol 추출물을 다시 $CHCl_3$, n-buthanol, $H_2O$ 분획으로 나누어 galactosamine 독성에 의한 간세포 보호작용을 재검색한 결과, 50 ppm의 농도에서 간세포 보호작용을 가지는 대조약물 silybin의 41.3% 보호효과에 비하여 각각 51.2%, 10.6%, 23.1%의 보호활성을 나타내었다. 이 중 가장 활성이 큰 $CHCl_3$, 분획을 가지고 활성의 추적 분리 방법으로 소분획 CH-II, V, VI의 활성 subfraction을 얻고 이를 다시 TLC와 활성검색을 병용하여 CH-(V+VI)-d, -e, -j의 sub-subfraction을 얻을 수 있었으며 이들은 50 ppm의 농도에서 각각 41.3%, 51.3%, 47.3%의 보호활성을 보였다. 활성 소분획 CH-II, V, VI는 모두 $[^3H]-uridine$ uptake 시험을 통한 RNA 생합성에는 영향을 미치지 않았으나 total GSH 값은 간세포 보호약물인 silymarin 대조구 $100\;{\mu}M$ 농도에서의 59.7% 수준의 회복효과에 비하여 각각 49.8%, 43.9%, 47.5%의 회복효과를 보였다. 또한 (reduced GSH)/(total GSH) 값도 silymarin $100\;{\mu}M$에서의 0.850에 비하여 각각 0.871, 0.863, 0.872로 유사한 수치를 타나내었다. 이 연구결과로 쑥갓의 간세포 보호작용을 처음으로 검색, 확인하였으며 컬럼 크로마토그래피를 이용하여 활성이 있는$CHCl_3$ 소분획을 분리하였으므로 이들 소분획으로 부터 더욱 활성물질을 순수분리하여 그 구조를 밝히고 간세포 보호활성의 기전에 대하여도 다양한 방향의 연구가 수행되어야 할 것이다.