• Animal Bioscience
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• 제34권6호
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• pp.957-966
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• 2021

Objective: Ovarian follicular development, which dependent on the proliferation and differentiation of granulosa cells (GCs), is a complex biological process in which miRNA plays an important role. Our previous study showed that miR-458b-5p is associated with ovarian follicular development in chicken. The detailed function and molecular mechanism of miR-458b-5p in GCs is unclear. Methods: The luciferase reporter assay was used to verify the targeting relationship between miR-458b-5p and catenin beta-1 (CTNNB1), which is an important transcriptional regulatory factor of the Wnt/β-catenin pathway. The cell counting kit-8 (CCK-8) assay, flow cytometry with propidium iodide (PI) and annexin V-fluorescein isothiocyanate (FITC) labeling were applied to explore the effect of miR-458b-5p on proliferation, cell cycle and apoptosis of chicken GCs. Quantitative real-time polymerase chain reaction and Western blot were used to detect the mRNA and protein expression levels. Results: We demonstrated that the expression of miR-458b-5p and CTNNB1 showed the opposite relationship in GCs and theca cells of hierarchical follicles. The luciferase reporter assay confirmed that CTNNB1 is the direct target of miR-458b-5p. Using CCK-8 assay and flow cytometry with PI and Annexin V-FITC labeling, we observed that transfection with the miR-458b-5p mimics significantly reduced proliferation and has no effects on apoptosis of chicken GCs. In addition, miR-458b-5p decreased the mRNA and protein expression of CD44 molecule and matrix metallopeptidase 7, which are the downstream effectors of CTNNB1 in Wnt/β-Catenin pathway and play functional roles in cell proliferation. Conclusion: Taken together, the data indicate that miR-458b-5p regulates ovarian GCs proliferation through Wnt/β-catenin signaling pathway by targeting CTNNB1, suggesting that miR-458b-5p and its target gene CTNNB1 may potentially play a role in chicken ovarian follicular development.

• 한국육종학회지
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• 제41권4호
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• pp.463-467
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• 2009

Allium 속 근연종인 파와 양파간 종간교잡을 이용하여 양파로부터 새로운 형질을 도입하는 과정에서 유래되는 종간교잡 F1과 여교잡 세대들의 게놈크기의 변화를 flow cytometry를 이용하여 측정한 결과 다음과 같은 결과를 얻었다. 1. 양파와 파의 종간교잡 F1의 식물학적 특성은 양친의 중간형을 보였으나 여교잡이 진전됨에 따라 반복친인 파의 표현형이 우세하였다. flow cytometry를 이용하여 2C nuclear DNA content을 측정한 결과 $F_1$은 양친에 비해 오히려 증가하였으나 여교잡이 진행됨에 따라 급속히 감소하여 $BC_2F_1$에서는 반복친과 비슷한 수준으로 감소하였다. 여교잡세대의 자식인 $BC_1F_2$은 반복친과 비슷한 $23.1{\pm}1.0pg$으로 측정되었으나 $BC_2F_2$는 $22.9{\pm}0.4pg$으로 반복친의 2C nuclear DNA content인 $23.2{\pm}0.2pg$ 보다 오히려 감소하였다.

• Journal of Nutrition and Health
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• 제52권2호
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• pp.157-167
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• 2019

본 연구에서는 ice plant의 ethanol 추출물과 분획물의 항산화 및 대장암세포 성장억제 활성을 in vitro 수준에서 평가하는 것을 목적으로 하였다. Ethanol 추출물의 총 폴리페놀 함량 (3.7 mg GAE/g), 총 카로티노이드 함량 ($13.2{\mu}g/g$), DPPH 라디칼 소거활성 (21.0%), 철 환원력 (21.0%)보다 butanol 분획물의 총 폴리페놀 함량 (5.4 mg GAE/g), 총 카로티노이드 함량 ($86.6{\mu}g/g$), DPPH 라디칼 소거활성 (34.9%), 철 환원력 (80.8%)이 더 높았다. 또한 HCT116 대장암세포에서 세포 내 활성산소종 수준을 감소시키거나 세포 성장을 억제하는데 있어서 ethanol 추출물보다 butanol 분획물의 활성이 더 컸다. 대장암세포의 성장을 억제하는데 있어서 butanol 분획물이 ethanol 추출물보다 더 효과적이었던 것은 butanol 분획물의 apoptosis 유도활성이 ethanol 추출물의 활성보다 더 컸고 butanol 분획물만이 G2/M기억류활성을 나타냈기 때문인 것으로 생각된다. 앞으로 이와 같은 결과를 초래하는 주요 활성성분을 분리 동정하고 ice plant의 항산화 활성 및 대장암세포 성장억제 효과가 in vivo 수준에서 재현되는지 검증하며 이와 관련된 세부기전을 탐색하는 심도 있는 연구가 필요할 것으로 생각된다.

• Tuberculosis and Respiratory Diseases
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• 제82권2호
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• pp.133-142
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• 2019

Background: Idiopathic pulmonary fibrosis involves irreversible alveolar destruction. Although alveolar epithelial type II cells are key functional participants within the lung parenchyma, how epithelial cells are affected upon bleomycin (BLM) exposure remains unknown. In this study, we determined whether BLM could induce cell cycle arrest via regulation of Schlafen (SLFN) family genes, a group of cell cycle regulators known to mediate growth-inhibitory responses and apoptosis in alveolar epithelial type II cells. Methods: Mouse AE II cell line MLE-12 were exposed to $1-10{\mu}g/mL$ BLM and $0.01-100{\mu}M$ baicalein (Bai), a G1/G2 cell cycle inhibitor, for 24 hours. Cell viability and levels of pro-inflammatory cytokines were analyzed by MTT and enzyme-linked immunosorbent assay, respectively. Apoptosis-related gene expression was evaluated by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Cellular morphology was determined after DAPI and Hoechst 33258 staining. To verify cell cycle arrest, propidium iodide (PI) staining was performed for MLE-12 after exposure to BLM. Results: BLM decreased the proliferation of MLE-12 cells. However, it significantly increased expression levels of interleukin 6, tumor necrosis factor ${\alpha}$, and transforming growth factor ${\beta}1$. Based on Hoechst 33258 staining, BLM induced condensation of nuclear and fragmentation. Based on DAPI and PI staining, BLM significantly increased the size of nuclei and induced G2/M phase cell cycle arrest. Results of qRT-PCR analysis revealed that BLM increased mRNA levels of BAX but decreased those of Bcl2. In addition, BLM/Bai increased mRNA levels of p53, p21, SLFN1, 2, 4 of Schlafen family. Conclusion: BLM exposure affects pulmonary epithelial type II cells, resulting in decreased proliferation possibly through apoptotic and cell cycle arrest associated signaling.

• 한국동물생명공학회지
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• 제35권4호
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• pp.297-306
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• 2020

The aims of the present study were to confirm that regulation of the PA and environment via TGF-β regulation of sperm by Percoll-separated in porcine uterine epithelial cells. And, it was performed to identify the cytokines (TGF-β1, 2 and 3, TGF-β receptor1 and 2; interleukin, IL-6, IL-8) and PA-related genes (urokinase-PA, uPA; tissue-PA, tPA; PA inhibitor, PAI; uPA-receptor, uPAR) by spermatozoa. The experiment used porcine uterus epithelial cells (pUECs) and uterine tissue epithelial cells, Boar sperm were separated by discontinuous Percoll density gradient (45/90%), and tissues were co-incubated with spermatozoa, followed by real-time PCR. PA activity was measured of sperm by discontinuous Percoll density gradient (45/90%) for 24 hours. To measure viability and acrosome damage of sperm double stained propidium iodide (PI) and SYBR-14 or FITC-PNA were used. In results, binding ratio of Percoll-separated sperm was found no differences, but sperms isolated from 90% Percoll layer reduced PA activity (p < 0.05). when co-cultured sperm selected Percoll in porcine uterus tissues epithelial cells, 90% layer sperm increased TGF-β R1, contrastively tPA and PAI-1 in comparison with control (p < 0.05). 45% sperm was decreased the expression of uPA (p < 0.05). TGF-β decreased PA activity in the supernatant collected from pUECs (p < 0.05). Especially, The group including uPA, PAI-1 were induce sperm intact, while it was reduced in sperm damage when compared to control (p < 0.05). Also, there was no significant difference group of tPA and tPA+I in the dead sperm and acrosome damage compared to control. The expression of tPA and PAI showed a common response. Percoll-separated spermatozoa in 90% layer reduced tPA and IL-related gene mRNA expression. Thus, Percoll-sparated sperm in 90% layer show that it can suppress inflammation through increased expression of TGF-β and downregulation of PA and IL in epithelial cells compared to 45% layer Percoll.

• The Korean Journal of Physiology and Pharmacology
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• 제26권6호
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• pp.427-438
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• 2022

Pyroptosis, a form of cell death associated with inflammation, is known to be involved in diabetic nephropathy (DN), and discoid domain receptor 1 (DDR1), an inflammatory regulatory protein, is reported to be associated with diabetes. However, the mechanism underlying DDR1 regulation and pyroptosis in DN remains unknown. We aimed to investigate the effect of DDR1 on renal tubular epithelial cell pyroptosis and the mechanism underlying DN. In this study, we used high glucose (HG)-treated HK-2 cells and rats with a single intraperitoneal injection of streptozotocin as DN models. Subsequently, the expression of pyroptosis-related proteins (cleaved caspase-1, GSDMD-N, Interleukin-1β [IL-1β], and interleukin-18 [IL-18]), DDR1, phosphorylated NF-κB (p-NF-κB), and NLR family pyrin domain-containing 3 (NLRP3) inflammasomes were determined through Western blotting. IL-1β and IL-18 levels were determined using ELISA. The rate of pyroptosis was assessed by propidium iodide (PI) staining. The results revealed upregulated expression of pyroptosisrelated proteins and increased concentration of IL-1β and IL-18, accompanied by DDR1, p-NF-κB, and NLRP3 upregulation in DN rat kidney tissues and HG-treated HK-2 cells. Moreover, DDR1 knockdown in the background of HG treatment resulted in inhibited expression of pyroptosis-related proteins and attenuation of IL-1β and IL-18 production and PI-positive cell frequency via the NF-κB/NLRP3 pathway in HK-2 cells. However, NLRP3 overexpression reversed the effect of DDR1 knockdown on pyroptosis. In conclusion, we demonstrated that DDR1 may be associated with pyroptosis, and DDR1 knockdown inhibited HG-induced renal tubular epithelial cell pyroptosis. The NF-κB/NLRP3 pathway is probably involved in the underlying mechanism of these findings.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.69-69
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• 2001

The cell cycle phase in which donor nuclei exist prior to nuclear transfer is an important factor governing developmental rates of reconstituted embryos. It was suggested that quiescent G0 and cycling G1 cells could support normal development of reconstituted embryos. In a quest of optimized donor nuclei treatment prior to nuclear transfer, this study was undertaken to examine the cell cycle characteristics of bovine fetal and adult somatic cells when cultured under a variety of culture treatments and the cell cycle change with the lapse of time after trypsinization. This was archived by measuring the DNA content of cells using flow cytometry, Cultured fetal fibroblast cells, adult skin and muscle cells, and cumulus cells were divided by 3 culture treatments; 1) grown to 60-70% confluency (cycling), 2) serum starved culture, 3) culture to confluency. Trypsinized cells were fixed by 70% ethanol and stained with propidium iodide. For one experiment, trypsinized cells were resuspended in DMEM＋10% FBS and incubated for 1.5, 3 and 6 h with occasional shaking before ethanol fixation. Cell cycle phases were determined by flow cytometry enabling calculation of percentages of G0＋G1, S and G2＋M. The majority of cells were in G0＋Gl stage regardless of origin of cells. Cultures that were serum starved or cultured to confluency contained significantly (P<0.05) higher percentages of cells in G0＋G1 (89.5-95.4%). For every cell lines and culture treatments, percentages of cells in existing in G0＋G1 increased with decreasing of the cell size from large to small. In the serum starved and confluency groups, about 98% of small cells were in G0＋G1 Serum starved culture contained higher percentages of small-sized cells (38.5-66.9%) than cycling and confluent cultures regardless of cell lines (P<0.05). After trypsinization of fetal fibroblast and adult skin cells that were serum starved and cultured to confluency, the percentages of cells in G0＋G1 significantly increased by incubation for 1.5(95.7-99.5%) and 3.0 h (95.9-98.6%). The results suggest that the efficient synchronization of bovine somatic cells in G0＋G1 for nuclear transfer can be established by incubation for a limited time period after trypsinization of serum starved or confluent cells.

• Journal of Veterinary Science
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• 제23권2호
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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.

• 한방안이비인후피부과학회지
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• 제37권1호
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• pp.1-16
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• 2024

Objectives : We aimed to study the effect of Smilax China L.(SCL), which has anti-inflammatory, antioxidant, and anticancer effects, on the growth of skin cancer cells. Methods : HaCaT cells, a normal human cell line, and skin cancer cells including A431, SK-MEL-5 and SK-MEL-28 cells were treated with Smilax China L. ethanol extract(SCL-EtOH) at concentrations of 5, 10, 20 and 40㎍/㎖. Meanwhile, JB6 Cl41, a normal mouse epithelial cell line, was treated with epidermal growth factor(EGF) and phorbol 12-myristate 13-acetate(TPA), an inflammatory factor, to induce cell transformation and treated with SCL-EtOH. In addition, we treated SK-MEL-5 and SK-MEL-28 cells with SCL-EtOH at various concentrations and checked the effect on the cell cycle. Results : As a result, it showed no toxicity to HaCaT cells up to the highest concentration of 40㎍/㎖, and significant cell growth inhibition to A431, SK-MEL-5 and SK-MEL-28 cells in a time- and concentration-dependent manner. In addition, as a result of checking the shape of skin cancer cells according to SCL-EtOH treatment, it was observed that as the concentration increased, the number of normally attached and growing cells decreased and the shape of the cells changed. Colony formation was significantly reduced in a concentration-dependent manner in JB6 Cl41 cells treated with EGF or TPA. Flow cytometry analysis with propidium iodide(PI) staining showed that SCL-EtOH induced the G2/M phase arrest. We further confirmed the decrease in Cyclin B1 expression and increase in p27 expression associated with the G2/M phase of the cell cycle through western blot analysis. Flow cytometry analysis confirmed that SCL-EtOH induced cell apoptosis. Furthermore, through Western blot analysis, it was observed that the expression of cleaved-caspase-7, which is related to apoptosis, increased. Finally, it was confirmed that the expression of COX-2, an inflammatory marker protein, decreased in a concentration-dependent manner with SCL-EtOH. Conclusions : Through the above results, we have established a basis for applying SCL to the treatment of skin cancer.

• Tuberculosis and Respiratory Diseases
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• 제56권2호
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• pp.187-197
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• 2004

연구배경 : Nitric Oxide(NO)는 매우 다양한 생물학적 조절기능을 수행하는 분자로서 심혈관계, 신경계, 면역기능 등에 관여함은 물론 최근 세포 사에도 직 간접적으로 영향을 미치고 있음이 알려져 있다. NO의 이렇게 복잡한 생물학적 기능 수행은 reactive oxygen species(ROS), metal ions 및 단백질 등과 복잡한 상호작용에 의한 것이며 NO가 나타내는 생물학적 효과는 용량-의존적이며, 세포-특이적이라고 밝혀져 있다. NO는 간세포 및 현관내피세포에서는 아포프토시스를 억제하지만 종양세포 및 신경세포 등에서는 아포프토시스를 유도하는 것으로 보고되고 있다. NO는 여러 호흡기질환의 병태생리에도 관여하는 것으로 알려져 있는바 천식과 같은 염증성 기도 질환에서 호기 NO가 증가되어있는 반면 흡연자나 일차성 폐 고혈압 환자에서는 감소되어 있다고 보고되고 있다. 이러한 배경에서 NO가 폐 상피 세포의 세포 사에 미치는 영향과 신호전달 경로를 밝히기 위하여 본 연구를 시행하였다. 방 법 : 폐 상피 세포로는 A549 세포 주를, NO donor로서는 SNAP (S-nitroso-N-acetyl-penicillamine)과 SNP(sodium nitroprusside)를 사용하였다. 세포 독성 검사는 crystal violet assay를 이용하였고 아포프토시스 assay는 Hoechst 33342와 propium iodide(PI) 이중 염색 후 형광현미경을 이용하여 핵의 형태학적변화를 관찰함으로써 괴사(necrosis)와 감별하였다. 철에 의한 NO 유도성 세포 사 억제 효과를 관찰하기 위하여 RBC와 FeSO4를 이용하였다. NO 유도성세포사의 신호전달 경로에 bcl-2와 p53이 미치는 영향을 평가하기 위하여 bcl-2 과 발현 세포 주 (A549-bcl-2)와 p53 knock out 세포 주 (A549-E6)를 대상으로 세포독성을 비교하였고 p53 활성화는 Western blot을 이용하여 확인하였다. 결 과 : A549 세포 주에서 SNAP과 SNP 모두 농도-의존적 세포독성을 관찰할 수 있었다. 아포프토시스 assay에서 SNAP은 저 농도에서는 아포프토시스를, 고농도에서는 괴사를 유도함을 관찰하였고 SNP는 농도에 상관없이 세포사가 괴사의 형태를 나타냄을 확인하였다. 이는 SNP가 순순한 NO donor가 아니라 cyanide에 의한 세포독성의 결과라고 생각되며 고농도의 SNAP에 의한 괴사 유도는 peroxynitrite 생성에 의한 결과임을 시사한다. SNAP에 의한 세포 사는 RBC와 FeSO4등 철에 의해 억제됨을 확인하였고 bcl-2에 의해서 억제되었으며 p53을 활성화시키고 p53 knock out에 의해 차단되었다. 결 론 : 폐상피세포에서 NO는 저 농도에서는 아포프토시스를 고농도에서는 괴사에 의한 세포 사를 유도하며 철이 중요한 억제제이며 bcl-2 및 p53이 신호전달 경로에 있어서 중요한 역할을 담당하는 것으로 생각된다.