• Toxicological Research
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• 제5권1호
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• pp.9-15
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• 1989

The effect of butylated hydroxytoluene (BHT) and its major metabolite, 3, 5-di-tert-butyl-4-hydroxybenzoic acid (BHT-acid) on the uptake of taurocholate into hepatocytes was studied using the primary culture of rat hepatocytes. Hepatocyte were isolated by an in situ collagenase perfusion technique and maintained as a monolayer in serum-free meadia for 24 hours before use. The uptake of taurocholate was saturable with an apparent Km of 12.8+2.8 MuM and Vmax of 0.18+0.01 nmol/mg/min. Both BHT and BHT-acid inhibited the hepatocellular uptake of taurocholate when they were added to the culture.

• 한국발생생물학회지:발생과생식
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• 제24권3호
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• pp.207-214
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• 2020

Primary cell culture is a sufficient method frequently used to study the cellular properties and mechanisms of isolated cells in a controlled environment. In this study, an embryonic cell line (FGBC8) derived from the blastula stages of embryos of olive flounder Paralichthys olivaceus was developed. Furthermore, conditions for optimal long-term maintenance of this primary embryonic cell culture were investigated. Morphologically, FGBC8 cells were composed primarily of epithelial-like cells. FGBC8 cells were subcultured for >160 passages over ~830 days. The doubling time of FGBC8 cells was 73.8 h, and the modal diploid chromosome number was 48. FGBC8 cells transfected with green fluorescence protein (GFP)-expression plasmid exhibited a strong signal 48 h after transfection. Consequently, we demonstrated that fish serum is a crucial supplement for the long-term survival and maintenance of comparable morphology in these primary embryonic cells. Our results can be used as a guide for primary embryonic cell cultures for other fish species and may be useful for cell biotechnological applications.

• 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.

• 대한수의학회지
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• 제36권3호
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• pp.551-563
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• 1996

This study investigated the properties of primary cultured proximal tubule cells in hormonally defined(insulin, transferrin, and hydrocortisone), serum-free medium or 10% serum-supplemented medium. The growth rate of the primary cultured proximal tubule cells was lower in the hormonally defined, serum-free medium than in the 10% serum- supplemented medium(p < 0.05), while the activities of brush border marker enzymes, alkaline phosphatase(AP), leucine aminopeptidase(LAP), and y-glutamyl transpeptidase(${\gamma}$-GTP) were increased(p < 0.05). The activities of these enzymes, however, decreased with the lapse of incubation time to 50-70% after 6 days culture compared to those of the freshly-prepared proximal tubules. The enzymatic activities of the primary cultured proximal tubul cells on 6, 9, 12, and 15 days of culture were significantly increased in the hormonally defined, serum-free medium compared to the 10% serum-supplemented medium(p < 0.05). The functional differentiation of the primary culture was examined by observing multicellular domes of the confluent monolayer, which is indicative of transepithelial solute transport. The dome formation by the proximal tubule cultures occurred at a higher frequency in the hormonally defined, serum-free medium than in the 10% serum-supplemented medium(p < 0.05). Upon electron microscopic examination, an increased density of the brush border was observed in the hormonally defined, serum-free medium compared to the cells grown in 10% serum-supplemented medium. The activities of $Na^+$glucose cotransporter($^{14}C$-a-MG uptake), $Na^+$phosphate cotransportere($^{32}P$ uptake) and $Na^+$ transporter($^{22}Na^+$ uptake) in the brush border membrane, and of $Na^+/K^+$-ATPase($^{86}Rb$ uptake) in the basolateral membrane were significantly stimulated in the hormonally defined, serum-free medium than in 10% serum-supplemented medium(p < 0.05). In conclusion, the primary cultured proximal tubule cells grown in the hormonally defined, serum-free medium demonstrated a slower growth rate, but the functions of cell were enhanced.

• 대한가정학회지
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• 제49권9호
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• pp.99-109
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• 2011

The purpose of this study was to characterize the new women, modern girls and modern boys from the 1920's to the 1930's as a modern new generation, the primary consumer of modern consumption culture, and to examine their values, lifestyles, consumption culture and clothing attitudes. The data were obtained from the magazines and newspapers published from 1920's to 1930's and previous literatures, and analyzed by qualitative content analysis. The results were as follows: A modern new generation meant the new women, modern girls, and modern boys seeking for the western looks and cultural tastes. The values of a new generation people were individualism, materialism, and modernism which was the same as Americanism. They enjoyed western lifestyles and sports and consumed new mass media and popular culture. Their clothing attitudes were fashion orientation, conformity, symbolism, conspicuous consumption, aesthetic value, individuality, and practicality.

• 대한바이러스학회지
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• 제28권4호
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• pp.317-325
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• 1998

The primary culture of human nasal epithelial cells was performed using the inferior nasal turbinate tissues, and infected with Hantaan virus to examine the hypothesis of airborne transmission of Hantaan virus in humans. The primary culture cells were identified as epithelial cells by morphologic and immunologic analyses. The viral antigens were detected in the primary human nasal epithelial cells infected with Hantaan virus by immunofluorescence staining. The ICAM-1 induction by Hantaan virus or $IFN-{\gamma}$ was examined in the primary human nasal epithelial cells and human lung fibroblasts (WI-38). Hantaan virus induced the surface ICAM-1 in WI-38 cells in a time-dependent manner, and $IFN-{\gamma}$ induced the surface ICAM-1 in a dose-dependent manner in HNEC and WI-38 cells. These results revealed that the human nasal epithelial cells are susceptible to Hantaan viral infection supporting the hypothesis of airborne transmission of Hantaan virus in humans. The human lung fibroblasts also might have an important role in the pathogenesis of Hantaan virus through the induction of ICAM-1.

• 한국수정란이식학회지
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• 제27권3호
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• pp.189-192
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• 2012

Although embryonic stem cells (ESCs) or ES-like cells are reported from many mammalian species other than the mouse, the culture system for murine ESCs may not be suitable to the other species. Previously many other research groups have modified either human or mouse ESC culture systems for bovine ESC culture. In this study, we compared three different culture mediums consisting of DMEM, ${\alpha}$-MEM or KnockOut$^{TM}$-DMEM (KO), which are modified from human or mouse ESC culture system, for the generation of bovine ESCs. In this study, some pre-requisite events which are important for establishment and long-term propagation of ESCs such as inner cell mass (ICM) attachment on feeder cells, primary colony formation and sustainability after passaging. Once the ICM clumps attached on feeder cells, this was designated as passage 0. In regards to the rate of ICM attachment, ${\alpha}$-MEM was superior to the other systems. For primary colony formation, there was no difference between DMEM and ${\alpha}$-MEM whereas KO showed lower formation rate than the other groups. For passaging, the colonies were split into 2~4 pieces and passed every 5~6 days. From passage 1 to passage 3, DMEM system seemed to be appropriate for maintaining putative bovine ESCs. On the other hand, ${\alpha}$-MEM tended to be more suitable after passage 6. Although ${\alpha}$-MEM support to maintain a ES-like cell progenies to passage 15, all three culture systems which are modified from human or mouse ESC culture media failed to retain the propagation and long-term culture of putative bovine ESCs. Our findings imply that more optimized alternative culture system is required for establishing bovine ESC lines.

• Pediatric Infection and Vaccine
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• 제3권2호
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• pp.123-127
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• 1996

Purpose : The most patients with acute streptococcal pharyngitis lack of classic clinical manifestations, therefore diagnostic laboratory test such as the throat culture or a rapid antigen detection test are frequently employed in primary practices of developed countries. We'd like to know the accuracy of the throat swab culture as gold standard for diagnosis of streptococcal infection with studying the discordant and concordant rate of duplicate culture. Methods : The study included 89 normal school children (boys:50, girls:39) who were attending Uljin primary school in Uljin, Kyong Sang Buk Do on March 1996. We obtained simultaneous 2 times of throat swab from each subject, and plating and streaking on 5-7% of sheep blood agar separately. We counted the characteristic beta-hemolytic colonies after overnight incubation. Results : 1) The carrier rate of beta-hemolytic streptococci at first culture is 25.1% and second one is 29.2%. 2) Ten out of 89(11.2%) is discordant in duplicate culture. 3) Culture containing less than 50 colonies of beta-hemolytic streptococci (+2) in first culture is 70.4%, second one is 85.7%. 4) Number of colonies is less than 50 in all ten discordant children. Conclusions : The discordant rate of duplicate throat swab cullture for beta-hemolytic streptococci is 11.2%, even if the subjects are normal school children. About 5% of individuals harboring beta-hemolytic streptococci in the pharynx may be missed by a single throat culture. If we are trying to examine the patients with pharyngitis, the discordant rate will be much lower than this results.

• 식물조직배양학회지
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• 제21권1호
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• pp.35-39
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• 1994

내냉성의 지표로 이용되는 적고의 발현정도가 상이한 벼품종 및 유성계통과 그들이 상호교잡된 F$_1$씨 청배양에서 최초 형성된 캘러스(primary callus)를 저온 스트레스 (stress) 처리하여 내냉성 세포의 기내선발 가능성을 검토하였다. 캘러스 형성율이나 저온 스트레스를 통한 식물체 분화율은 자포니카형이나 자포니카형/통일형 조합이 통일형이나 통일형간 조합보다 높았으며 저온 스트레스 처리일수의 증가에 따라 백색체 분화율이 증가하는 경향을 보였다. 약배양 효율에 있어서 상호교잡에 따른 세질효과는 나타나지 많았다. 고정도가 높은 품종이나 교잡 F$_1$의 약배양 후대 계통에서 캘러스의 저온 stress처리일수에 따른 적고 발현정도의 변이가 인정되지 많았다. 적고발현 정도가 상이한 품종간 교잡 F$_1$에서 유래된 연배양 후대 계통의 적고 발현은 양친 범위 내에 대부분이 분포하였으나 적고 발현이 낮은 방향의 초월분리계통도 나타났다. 최초 형성된 캘러스의 저온 stress 처리($0^{\circ}C$ 10일)에서 분화된 약배양 계통, 무처리에서 분화된 약배양 계통과 F$_2$ 집단간에는 적고 배양의 빈도 분포에서 차이가 인정되지 않았다. 따라서 벼 약배양에서 최초 형성된 캘러스 수준에서 저온 스트레스를 통해 내냉성 세포를 직접적인 기내선발방법은 효과적이지 못함이 밝혀졌다.

• 한국가금학회지
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• 제23권3호
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• pp.113-119
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• 1996

A series of experiments were conducted to investigate the role of protein kinase C (PKC) as a second messenger in vasoactive intestinal peptide (VIP) mediated prolactin secretion. Primary pituitary cells (106 cells/treatment) were separated from laying hens and incubated in M-199 with 5% chicken serum and 5% fetal calf serum. The VIP(0.1 $\pi$M) treatment enhanced prolactin Secretion into media upto 9-fold during 48-h incubation. The phorbol 12-myristate 13-acetate (PMA), a PKG agonist, increased prolactin secretion upto 2-fold at 0.1 nM PMA (P<0.01), and the prolactin secretion was not significantly higher than this concentration. Staurosporine (ST; 1.0$\pi$M) a PKC antagonist, decreased by 70% of 0.1 $\pi$M VIP-stimulated prolactin secretion and by 48% of 10 ${\mu}$M PMA-stimulated prolactin secretion (P<0.01). However, pituitary cell prolactin content did not differ in any treatment (P>0.05). In conclusion, these results indicate that the PKC second messenger system is involved in VIP-stimulated prolactin release in chicken primary pituitary cell culture.