• Animal Bioscience
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• v.35 no.11
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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.

• Journal of Veterinary Science
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• v.23 no.2
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• pp.40.1-40.13
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• 2022

Background: Somatic cell nuclear transfer (SCNT) is used widely in cloning, stem cell research, and regenerative medicine. The type of donor cells is a key factor affecting the SCNT efficiency. Objectives: This study examined whether urine-derived somatic cells could be used as donors for SCNT in pigs. Methods: The viability of cells isolated from urine was assessed using trypan blue and propidium iodide staining. The H3K9me3/H3K27me3 level of the cells was analyzed by immunofluorescence. The in vitro developmental ability of SCNT embryos was evaluated by the blastocyst rate and the expression levels of the core pluripotency factor. Blastocyst cell apoptosis was examined using a terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. The in vivo developmental ability of SCNT embryos was evaluated after embryo transfer. Results: Most sow urine-derived cells were viable and could be cultured and propagated easily. On the other hand, most of the somatic cells isolated from the boar urine exhibited poor cellular activity. The in vitro development efficiency between the embryos produced by SCNT using porcine embryonic fibroblasts (PEFs) and urine-derived cells were similar. Moreover, The H3K9me3 in SCNT embryos produced from sow urine-derived cells and PEFs at the four-cell stage showed similar intensity. The levels of Oct4, Nanog, and Sox2 expression in blastocysts were similar in the two groups. Furthermore, there is a similar apoptotic level of cloned embryos produced by the two types of cells. Finally, the full-term development ability of the cloned embryos was evaluated, and the cloned fetuses from the urine-derived cells showed absorption. Conclusions: Sow urine-derived cells could be used to produce SCNT embryos.

• Clinical and Experimental Pediatrics
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• v.52 no.2
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• pp.213-219
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• 2009

Purpose:Iron is a critical nutritional element that is essential for a variety of important biological processes, including cell growth and differentiation, electron transfer reactions, and oxygen transport, activation, and detoxification. Iron is also required for neoplastic cell growth due to its catalytic effects on the formation of hydroxyl radicals, suppression of host defense cell activities, and promotion of cancer cell multiplication. Chronic transfusion-dependent patients receiving chemotherapy may have iron overload, which requires iron-chelating therapy. We performed this study to demonstrate whether the iron chelating agent deferoxamine induces apoptosis in Saos-2 osteosarcoma cells, and to investigate the underlying apoptotic mechanism. Methods:To analyze the apoptotic effects of an iron chelator, cultured Saos-2 cells were treated with deferoxamine. We analyzed cell survival by trypan blue and crystal violet analysis, apoptosis by nuclear condensation, DNA fragmentation, and cell cycle analysis, and the expression of apoptotic related proteins by Western immunoblot analysis. Results:Deferoxamine inhibited the growth of Saos-2 cell in a time- and dose-dependent manner. The major mechanism for growth inhibition with the deferoxamine treatment was by the induction of apoptosis, which was supported by nuclear staining, DNA fragmentation analysis, and flow cytometric analysis. Furthermore, bcl-2 expression decreased, while bax, caspase-3, caspase-9, and PARP expression increased in Saos-2 cells treated with deferoxamine. Conclusion:These results demonstrated that the iron chelating agent deferoxamine induced growth inhibition and mitochondrial-dependent apoptosis in osteosarcoma Saos-2 cells, suggesting that iron chelating agents used in controlling neoplastic cell fate can be potentially developed as an adjuvant agent enhancing the anti-tumor effect for the treatment of osteosarcoma.

• Journal of Life Science
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• v.22 no.1
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• pp.16-24
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• 2012

Polyamines are essential for cell growth and differentiation; however their precise roles are unclear yet. In the present study, the cytotoxic effect of spermine (spm) on MCF-7 cells was investigated. In the MTT assay of MCF-7 cells treated with spm, cell viability was significantly decreased in a time-and dose-dependent manner. Cell viability measurement was confirmed by trypan blue staining. FACS analysis shows that sub-G1 was increased in a time-and dose-dependent manner too. When the cells were treated with spm, cells started to show morphological changes within 2 hrs. The expression of adhesion proteins (FAK and integrin ${\beta}1$), and cytoskeletal protein (actin) was checked by Western blotting analysis. Integrin ${\beta}1$ levels were slightly decreased, and FAK and actin levels were rapidly decreased with spm treatment. In confocal laser scanning microscopy, the distribution of actin did not change but the expression decreased in a dose-dependent manner with spm treatment. FAK was evenly distributed under the plasma membrane in the untreated control. However, at 10 ${\mu}M$ spm FAK seemed to move toward the cell nucleus. Integrin ${\beta}1$, which was mainly found in the focal point of the plasma membrane in the untreated control, dispersed through the entire plasma membrane in spm treatment. The present results indicate that cytotoxic effects of spm are triggered by the disruption of adhesion proteins and cytoskeletal protein.

• Reproductive and Developmental Biology
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• v.36 no.1
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• pp.65-70
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• 2012

For reconstituting genetic resource(Korean Native Chicken: KNC) with grem-line chimeric chicken made with cryopreserved biastdermal cells, the experiments were carried out to optimize cryopreservating conditions. Stage X biastdemal cells were collected from KNC embryos and dissociated. Cells were susupended in medium containing cyopretectant and fetal bovine serum(FBS), and distributed into plastic ampules. Cell susupensions were seeded to induce ice formation at $-7^{\circ}C$ to $-35^{\circ}C$ at in the experiments, the effect of modification of dissociation way, concentration of FBS and cell density on the vaibility of frezen-thawed cells were investigated by trypan blue exclusion. Then change the way of cell dissociation from pipetting to short time vortexing, viability of frozen-thawed cell tended to be increaced from 29 % to 52 %. Increase concentraition of FBS in frozen medium from 20 % to 80 % made viability of thawed cell from 28 % to 35 %. The viability of thawed cells were 33.9% frozen at 2 embryos/0.5 ml, and 43.6 % frozen at 20 embryos/0.5 ml. Furthermore, combination of three modifications make big improvement. The viability of frozen-thawed cell was 60 % for combinated method, and 41 % for general method. This result means the advance to practical cryoreservation of blastdermal cell of the KNC(Ogolgye breed).

• Journal of Veterinary Clinics
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• v.20 no.1
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• pp.1-6
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• 2003

Immunoenhancing effects of conjugated linoleic acid (CLA) isomers (l0t-l2c CLA, 9c-11t CLA, CLA mixture, 9c-11c CLA and 9t-11t CLA) on chemotactic activity of porcine peripheral blood polymorphonuclear cells (PMN) were examined. The chemotactic activity of PMN was evaluated by a modified Boyden chamber assay. CLA isomers at higher concentration of 50 to 200$\mu$M exhibited a low viability of cells by trypan blue exclusion. CLA isomers were used at concentration of 20uM showing no cytotoxic effect and high cell viability. CLA isomers themselves were not active or slight chemotactic for PMN. But culture supernatant from mononuclear cells (MNC) treated with 10t-12c CLA, 9c-11t CLA and CLA mixture except for 9c-11c. CLA and 9t-11t CLA enhanced remarkably chemotactic activity or porcine PMN PMN migration by culture supernatant from MNC treated with CLA mixture was found to be true chemotaxis by checkboard assay. This migration was also induced by porcine recombinant interleukin (rIL)-8. PMN chemotaxis caused both culture supernatant from MNC treated with CLA mixture and porcine rIL-8 was inhibited in a dose-dependent manner by addition of anti-porcine IL-8 polyclonal antibody. Therefore, these results strongly suggested that CLA (10t-12c CLA, 9c-11t CLA and CLA mixture) could stimulate porcine MNC to release and IL-8 like chemotactic activity.

• Korean Journal of Environmental Biology
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• v.27 no.1
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• pp.66-72
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• 2009

Diabetic Retinopathy (DR) is a leading cause of blindness among adults in the western countries. Hyperglycemia is a condition, that induces apoptotic cell death in a variety of cell types in diabetes, but the mechanism remains unclear. The aim of the study is to understand the effects of high Glucose on Human Retinal Endothelial Cells. Retinal endothelial cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM) containing 5, 25 and 50 mM Glucose, incubated for 24, 36 and 48 hours in humidified 5 % CO$_2$ incubator at 37$^{\circ}C$. Human Retinal Endothelial Cell Line (HREC) were characterized for morphology with different treatment by phase contrast microscopic analysis. Number of dead and viable cells was counted by trypan blue exclusion and supported by MTT assay. The intracellular Hydrogen peroxide (H$_2$O$_2$), a Reactive Oxygen Species (ROS) generation in high glucose conditions was assessed by FOX II assay and apoptosis by caspase-3 assay. The high glucose treated cells undergoing DNA fragmentation was witnessed by Agarose gel electrophoresis. We found that the cells incubated with 25 and 50 mM glucose containing medium for 48 hours altered the morphology of the cell, induced apoptosis and DNA fragmentation. The dead cell number were high in 25 and 50 mM when compared to the cells incubated with 5 mM glucose for 24, 36, and 48 hours. Also, the H$_2$O$_2$ levels and the activity of caspase-3 were increased in high glucose treated cells. Conclusions/interpretation: Our results demonstrated that elevated glucose induces apoptosis in cultured HREC. The hyperglycemia-induced increase in apoptosis may be dependent on caspase activation. The association between ROS generation and caspase-3 activation on high glucose treated cells is yet to be investigated.

• THE JOURNAL OF KOREAN ORIENTAL ONCOLOGY
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• v.4 no.1
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• pp.37-53
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• 1998

The sprig of Jinryungtang Gagambang has been used for curing as a traditional medicine without any experimental evidence to support the rational basis for their clinical use. This experiment was carried out to evaluate the possible therapeutic or antitumoral effects of Jinryungtang Gagambang extract against cancer, and to study some mechanisms responsible for its effect. The cytotoxic and antitumor effects were evaluated on human cell liens (A549, hep3B, Caki-1, Sarcoma 180) after exposure to Jinryungtang Gagambang extract using in ILS, colony forming efficency and SRB assay which were regarded as a valuable method for cytotoxic and antitumor effects of unknown compound on tumor cell lines. The results obtained in this studies were as follows. 1. As a result of exposure to Jinryungtang Gagambang extract, the proliferation of A549, hep3B, Caki-1, good correlations were shown from the results of SRB assay and those of clogenetic assay. 2. The oral administration of Jinryungtang Gagambang extract showed significant effects of increase of MST(mean survival time) and ILS(increased life span) depending on the increasing concentration. 3. Against squamous cell carcinoma induced by MCA, Jinryungtang Gagambang decreased not only the frequency of tumor production but also the number and weight of tumors per tumor bearing mice(TBM). Jinryungtang Gagambang also significantly suppressed the development of 3LL cell-implanted tumors by frequency and their size, and some developed tumors were regressed by the continuous treatment of Jinryungtang Gagambang extract into TBM. 4. Jinryungtang Gagambang extract also increased NK cell activities. According to the above results, it could be suggested that Jinryungtang Gagambang extract has prominent antiutmor effect.