• 제목/요약/키워드: hepatocyte primary culture

검색결과 51건 처리시간 0.025초

명태(Gadus chalcogrammus)의 일차 간세포 배양에서 온도 스트레스에 따른 HSP70 mRNA와 단백질 발현 (Expression of HSP70 mRNA and Protein based on the Thermal Stress in the Primary Hepatocyte Culture of Walleye Pollock (Gadus chalcogrammus))

  • 김소선;이창주;박장수
    • 한국환경과학회지
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    • 제29권6호
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    • pp.633-641
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    • 2020
  • Water temperature is one of the most important factors of fish survival, affecting the habitat, migration route, development, and reproduction. This experiment studied the induction level of heat shock protein (HSP70) mRNA and protein in a walleye pollock (Gadus chalcogrammus) primary hepatocyte culture based on different temperatures. Hepatocytes were attached at 7.5℃ for 24 hours. Hsp70 induction levels were then measured for 48 hours at 5, 8, 11, 14, and 17℃. The induction level was lowest at 5℃ and generally increased with temperature until 14℃. The induction level was reduced at 17℃, indicating that 14℃ is the highest tolerable temperature for hepatocytes. These data indicate that primary hepatocyte cell culture is under no stress at 5 and 8℃. Temperatures greater than 11℃ induce stress, showing similar induction patterns in both mRNA and protein in hepatocytes. The results suggest that 14℃ is the maximum internal defense temperature of walleye pollock survival.

Rat Primary Hepatocyte의 2차원 배양과 3차원 배양에 따른 생리 활성능과 대사능에 관한 연구 (Evaluation of primary hepatocyte function using 2D or 3D culture method for primary rat hepatocytes)

  • 임맑음;김영지;신유리안나;오건봉;황성수;김영임;허태영;옥선아
    • 한국수정란이식학회지
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    • 제31권3호
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    • pp.169-177
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    • 2016
  • 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 $3.5{\times}10^6$ 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.

Vitellogenin and Its mRNA Induction by $Estradiol-17\beta$ in the Primary Culture of Hepatocytes in the Rainbow Trout, Oncorhynchus mykiss

  • Hwang Un-Gi
    • Fisheries and Aquatic Sciences
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    • 제4권4호
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    • pp.186-191
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    • 2001
  • Vitellogenin (VTG) and VTG mRNA induction by $estradiol-17\beta\;(E_2)$ were examined in the primary cultures of hepatocyte in the rainbow trout. Hepatocytes were precultured for 2 days, then $E_2$ was added and cultured for another 5 days. Media and hepatocytes were then analyzed by electrophoresis and Northern blotting for VTG and VTG mRNA, respectively. The hepatocytes were formed a few aggregates within 5 days without further spreading to a monolayer. Cell viability and high DNA content were maintained during the incubation. The hepatocyte culture with E2 induced a weak VTG band at a molecular weight of 175kDa on Day 2 after $E_2$ addition. The relative amount of VTG was expressed in percentage of total protein concentrations. VTG was gradually increased as $1.9\%$ on Day 2, $6.3\%$ on Day 4 and $7.3\%$ on Day 5. VTG mRNA band was detected at about 6.6 kb in the culture with $E_2$ at day 1 of culture. The level of VTG mRNA expression linearly increased with time until Day 5 (r=0.97).

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A cost-effective and simple culture method for primary hepatocytes

  • Adaya, Sezin;Hasircib, Nesrin;Gurhana, Ismet Deliloglu
    • Animal cells and systems
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    • 제15권1호
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    • pp.19-27
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    • 2011
  • Hepatocytes, the major epithelial cells of the liver, maintain their morphology in culture dishes coated with extracellular matrix (ECM) components such as collagen and fibronectin or biodegradable polymers (e.g. chitosan, gelatin). In these coated dishes, survival of cells and maintaining of liver-specific functions may increase. The aim of this study was to determine a suitable, cost-effective and simple system for hepatocyte isolation and culture which may be useful for various applications such as in vitro toxicology studies, hepatocyte transplantation and bioartificial liver (BAL) systems. In order to obtain primary cultures, hepatocytes were isolated from liver by an enzymatic method and cultured on plates coated with collagen, chitosan or gelatin. Collagen, gelatin-sandwich and gelatin-cell mixture methods were also evaluated. Morphology and attachment of the cells were observed by inverted microscope and scanning electron microscope (SEM). An MTT assay was used to determine cell viability and mitochondrial activity.

Effect of Extracellular Calcium on Vitellogenin Production in the Culture of Hepatocytes in the Rainbow Trout, Oncorhynchus mykiss

  • Yeo In-Kyu;Mugiya Yasuo;Chang Young Jin;Hur Sung Bum;Yoo Sung Kyu
    • Fisheries and Aquatic Sciences
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    • 제1권1호
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    • pp.24-29
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    • 1998
  • Effect of extracellular calcium in vitellogenin (VTG) production in response to estradiol-17 $\beta$ $(E_2,\;2\times10^{-6}M)$ was examined in primary hepatocyte culture of rainbow trout, Onchorhynchus mykiss. Total calcium in estrogenized sera significantly increased, compared with the control, while diffusible calcium was insignificant. However, diffusible calcium in the incubation medium with $E_2$ was significantly reduced, compared with the control. The uptake of extracellular calcium by cultured hepatocytes signifIcantly increased 90 min after $E_2$ addition. Moreover, the accumulation of intracellular calcium increased in the cultures with $E_2$, regardless of the calcium concentrations in the incubation media. In addition, $E_2-primed $ VTG production was significantly decreased by withdrawal of E_2$ from the incubation medium. Moreover, VTG production by $E_2-primed$ hepatocytes was reduced by removing calcium from the incubation medium with or without $E_2$. These results suggest that the entry of extracellular calcium into the cytoplasm is an important step for VTG production in primary hepatocyte cultures in rainbow trout.

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Stimulatory Effects of cyclic AMP on Vitellogenin Induction by Estradiol-17$\beta$ in the Primary Culture of Hepatocytes in the Rainbow Trout Oncorhynchus mykiss

  • Yeo In-Kyu
    • Fisheries and Aquatic Sciences
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    • 제1권2호
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    • pp.153-158
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    • 1998
  • Effects of cyclic (c) AMP and G-protein related reagents (3-isobutyl-l-methyxanthine (IBMX), Forskolin (FSK), cholera toxin (CTX), and pertussis toxin (PTX≫ on estradiol-17$\beta$ induced vitellogenin (VTG) induction were examined in primary hepatocyte cultures in rainbow trout Oncorhynchus mykiss. The addition of IBMX, FSK, or CTX to the incubation medium markedly increased VTG production, while PTX was not effective in stimulating the production. It is well known that cAMP regulates phosphorylation and dephosphorylation through mediation of protein kinase A. These results suggest that VTG production is highly dependent on cAMP state in hepatocytes because of its highly phosphorylated nature.

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INDUCTION OF CYTOCHROME P-450 ASSOCIATED MONOOXYGENASE ACTIVITIES BY PHENOBARBITAL AND 3-METHYLCHOLANTHRENE IN PRIMARY CULTURES OF ADULT RAT HEPATOCYTES

  • Park, Seong-Kyu;Ha, Jong-Ryul;Kim, H.M.;Yang, K.H.
    • Toxicological Research
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    • 제3권1호
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    • pp.1-8
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    • 1987
  • In vitro induction of cytochrome 450 associated monooxygenase activities by phenobarbital (PB) and 3-methylcholanthrene (MC) was investigated in primary cultures of adult rat hepatocytes. PB and MC were added to the culture 24 hr after the initial plating of hepatocytes. A signiftcant increase of the activities of 7-ethoxycoumarin 0-deethylase and aryl hydrocarbon hydroxylase were observed in MC and PB treated culture. MC caused about 500% induction of the initial oxidation rates of both enzymes in 48 hr. However the PB maintained both enzyme activities close to the level of freshly isolated hepatocytes. Biphenyl 4-hydroxylase and aminopyrine N-demethylase activities were also induced by MC and PB. But the level of induction was less than that occuring with 7-ethoxycoumarin 0-deethylase and aryl hydrocarbon hydroxylase. When aflatoxin $B_1$ was added to the hepatocyte cultures which have been treated with MC or PB, it caused a significant increase of the unscheduled DNA synthesis at higher dose of aflatoxin $B_1$ as compared to those of untreated control hepatocyte cultures. The results suggest that microsomal enzyme activities can be selectively controlled preferably in hepatocyte cultures by the in vitro induction method. This principle may be useful for studying the metabolism and other toxicological studies.

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Severe choline deficiency induces alternative splicing aberrance in optimized duck primary hepatocyte cultures

  • Zhao, Lulu;Cai, Hongying;Wu, Yongbao;Tian, Changfu;Wen, Zhiguo;Yang, Peilong
    • Animal Bioscience
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    • 제35권11호
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    • pp.1787-1799
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    • 2022
  • Objective: Choline deficiency, one main trigger for nonalcoholic fatty liver disease (NAFLD), is closely related to lipid metabolism disorder. Previous study in a choline-deficient model has largely focused on gene expression rather than gene structure, especially sparse are studies regarding to alternative splicing (AS). In modern life science research, primary hepatocytes culture technology facilitates such studies, which can accurately imitate liver activity in vitro and show unique superiority. Whereas limitations to traditional hepatocytes culture technology exist in terms of efficiency and operability. This study pursued an optimization culture method for duck primary hepatocytes to explore AS in choline-deficient model. Methods: We performed an optimization culture method for duck primary hepatocytes with multi-step digestion procedure from Pekin duck embryos. Subsequently a NAFLD model was constructed with choline-free medium. RNA-seq and further analysis by rMATS were performed to identify AS events alterations in choline-deficency duck primary hepatocytes. Results: The results showed E13 (embryonic day 13) to E15 is suitable to obtain hepatocytes, and the viability reached over 95% by trypan blue exclusion assay. Primary hepatocyte retained their biological function as well identified by Periodic Acid-Schiff staining method and Glucose-6-phosphate dehydrogenase activity assay, respectively. Meanwhile, genes of alb and afp and specific protein of albumin were detected to verify cultured hepatocytes. Immunofluorescence was used to evaluate purity of hepatocytes, presenting up to 90%. On this base, choline-deficient model was constructed and displayed significantly increase of intracellular triglyceride and cholesterol as reported previously. Intriguingly, our data suggested that AS events in choline-deficient model were implicated in pivotal biological processes as an aberrant transcriptional regulator, of which 16 genes were involved in lipid metabolism and highly enriched in glycerophospholipid metabolism. Conclusion: An effective and rapid protocol for obtaining duck primary hepatocytes was established, by which our findings manifested choline deficiency could induce the accumulation of lipid and result in aberrant AS events in hepatocytes, providing a novel insight into various AS in the metabolism role of choline.

폴리우레탄 폼을 이용한 쥐 일차 간세포의 구상체 배양 (Formation of Spheroids of Adult Rat Primary Hepatocytes in Polyurethane Foam)

  • 안재일;이두훈
    • 대한의용생체공학회:의공학회지
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    • 제19권3호
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    • pp.215-224
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    • 1998
  • 본 논문은 급성 간 부전 환자나 간이식 대기상태에 있는 환자에 이용하기 위한 체외 간 보조장치를 개발하기 위한 기초 실험에 관한 것이다. 밀도 33kg/m, 평균 직경 500um, 60-70%의 window를 가진 폴리우레탄 폼을 15% NCO-프리폴리머로 제조하였다. 폴리우레탄 폼에 접종된 간세포는 3일 후에 거의 모두 100-150um 크기의 구상체를 형성하였다. BSA 코팅을 한 폴리우레탄 폼은 BSA로 코팅하지 않은 폴리우레탄 폼보다 간세포 구상체 형성 시간이 더 짧았다. 폴리우레탄 폼에 형성된 간세포 구상체의 간 기능을 증명하기 위하여 암모니아 제거율과 요소 및 알부민 분비율을 측정하였다. 폴리우레탄 폼은 간세포 구상체 배양에 적합하며, 폴리우레탄 폼 내에서의 간세포 구상체 배양은 체외 간 보조장치로의 개발 가능성이 높다.

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무지개송어의 간세포 초대배양에 의한 Vitellogeinin 합성 유도 (Vitellogenin Induction by Rainbow trout (Oncorhynchus mykiss) Hepatocytes in Primary Culture)

  • 여인규
    • 한국양식학회지
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    • 제11권4호
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    • pp.557-564
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    • 1998
  • 무지개송어의 초대배양 간세포에 있어서 Vitellogenin (VTG)의 합성 유도는 전기영동적 수법에 의해 행하였다. 간세포는 7일 동안 phositively charged dish를 이용한 배양으로 단층 확산되었다. 배양 7일의 간세포 생존율은 $E_2$의 유무에 따라 각각 20.7% 및 23.6%가 감소하였으며, DNA함유량도 각각 13.7% 및 14.0%가 감소하였다. $E_2$ 첨가군과 대조군간에는 생존율과 DNA 함유량에 있어서는 유의한 차이가 나타나지 않았다. 그리고, 총 단백질에 대한 VTG의 비율은 $E_2$$10^{-6}$ M의 첨가시에 최대치를 나타내었다. 그러나, 고농도($10^{-5}$M)에서는 오히려 감소하는 경향을 나타내었다.

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