• Clinical and Experimental Reproductive Medicine
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• 제32권2호
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• pp.133-147
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• 2005

Objectives: This study was carried out to establish human embryonic stem cells derived from frozen-thawed embryos using mouse embryonic fibroblasts (mEFs), human fetal skin and muscle fibroblasts as feeder cells, and to identify the characteristic of embryonic stem cells. Methods: When primary mEFs, human fetal skin and muscle fibroblasts were prepared, passaging on 4 days from replating could have effective trypsinization and clear feeder layers. Eight of 23 frozenthawed 4~8 cell stage embryos donated from consenting couples developed to blastocysts. Inner cell mass (ICM) was isolated by immunosurgery. ICM was co-cultured on mEFs, human fetal skin or muscle fibroblasts. The ICM colonies grown on mEFs, human fetal skin or muscle fibroblasts were tested the expression of stage specific embryonic antigen-3, -4 (SSEA-3, -4), octamer binding transcription factor-4 mRNA (Oct-4) and alkaline phosphatase surface marker. Results: We obtained 1 ICM colony from 2 ICM co-cultured on mEFs as feeder cells and did not obtain any ICM colony from 6 ICM clumps co-cultured on human fetal skin or muscle fibroblasts. The colony formed on mEFs could be passaged 30 times every 5 days with sustaining undifferentiated colony appearance. When the colonies cultured on mEFs were grown on human fetal skin or muscle fibroblasts, the colonies could be passaged 15 times every 9 days with sustaining undifferentiated colony appearance. The colonies grown on mEFs and human fetal fibroblasts expressed SSEA-4 and alkaline phosphatase surface markers and positive for the expression of Oct-4 by reverse transcription-polymerase chain reaction (RT-PCR). The produced embryoid body differentiated spontaneously to neural progenitorlike cells, neuron-like cells and beating cardiomyocyte-like cells, and frozen-thawed embryonic stem cells displayed normal 46,XX karyotype. Conclusions: The human embryonic stem cells can be established by using mEFs and human fetal fibroblasts produced in laboratory as feeder cells.

• Clinical and Experimental Reproductive Medicine
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• 제32권3호
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• pp.269-277
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• 2005

Objectives: Previously, we sought to compile a list of genes expressed during early folliculogenesis by using cDNA microarray to investigate follicular gene expression and changes during primordialprimary follicle transition and development of secondary follicles (Yoon et al., 2005). Among those genes, a group of genes related to the cell size growth was characterized during the ovarian development in the present study. Methods: We determined ovarian expression pattern of six genes related to the cell size growth (cyr61, emp1, fhl1, socs2, wig1 and wisp1) and extended into CCN family (${\underline{c}}onnective$ tissue growth factor/${\underline{c}}ysteine$-rich 61/${\underline{n}}ephroblastoma$-overexpressed), ctgf, nov, wisp2, wisp3, including cyr61 and wisp1 genes. Expression of mRNA and protein according to the ovarian developmental stage was evaluated by in situ hybridization, and/or semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), and immunohistochemistry, respectively. Results: Among 6 genes related to the cell size growth, cyr61 and wisp1 mRNA was detected only in oocytes in the postnatal day5 mouse ovaries. cyr61 mRNA expression was limited to the nucleolus of oocytes, while wisp1 was expressed in the cytoplasm and nucleolus of oocytes, except nucleus. cyr61 mRNA expression, however, was found in granulosa cells from secondary follicles. The rest 4 genes in the cell size growth group were detected in oocytes, granulosa and theca cells. Cyr61 and Wisp1 proteins were expressed in the oocyte cytoplasm from primordial follicle stage. Especially, Cyr61 protein was detected in pre-granulosa cells, Wisp1 protein was not. By using RT-PCR, we evaluated and decided that Cyr61 protein is produced by their own mRNA in pre-granulosa cells that was not detected by in situ hybridization. cyr61 and wisp1 genes are happen to be the CCN family members. The other members of CCN family were also studied, but their expression was detected in oocytes, granulose and theca cells. Conclusions: We firstly characterized the ovarian expression of genes related to the cell size growth and CCN family according to the early folliculogenesis. Cyr61 protein expression in the pre-granulosa cells is profound in meaning. Further functional analysis for cyr61 in early folliculogenesis is under investigation.

• 한방안이비인후피부과학회지
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• 제31권1호
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• pp.52-70
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• 2018

Objectives : Atopic dermatitis(AD) and environmental factors are closely related, but there is lack of oriental medical examination. So we compared the relationship between AD and various environmental factors in Oriental medicine and Western medicine. Methods : We described the relationship between AD and environmental factors through the latest papers and a review of the oriental medicine literature. Results : The regional diversity of AD incidence implies a close relationship between climate factors and AD, and high altitude and low pH springs also have an effect on AD. Air pollutants from industrialization and urbanization aggravate AD. The increase in indoor residence time and the increase in room temperature and humidity have also increased the sensitization to allergens such as house dust mite. In oriental medicine, wind(風) is one of the environmental factors and is an inflammatory state due to external irritation. Wind-Humidity(風濕) refers to erythematous wetting dermatitis with itching and exudation, Wind-Fever(風熱) refers to acute inflammatory reaction with erythematous papules and plague, and Blood-Weakness(血虛) refers to aggravation and chronicization of inflammation due to persistence of skin barrier impairment. Conclusions : We examined the relationship between AD and various environmental factors. We also described the oriental medical viewpoints of the environmental factors in the occurrence of AD and skin barrier impairment.

• 한국식품저장유통학회지
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• 제18권3호
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• pp.414-422
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• 2011

자색 고구마로부터 페놀성 화합물 추출 최적 조건은 50% 에탄올로 12시간 추출하였을 때 최대 용출량을 나타내었다. 불과 50% 에탄올을 용매로 하여 제조한 자색 고구마 종류별 추출물의 항산화 활성 측정 결과 전자공여능, ABTS, PF 측정에서 일반 고구마에 비하여 자색 고구마 자미, 신자미, 연자미 품종 모두 높게 나타났으며, TBARs 측정결과 모든 추출물에서 30% 이하로 낮은 TBARs 생성 억제활성을 나타내었다. 피부상재균에 대한 최소저해농도(MIC)를 측정한 결과 Staphylococous aureus 및 Escherichia coli에 대한 MIC는 자미, 연자미, 신자미에서 각각 5,000 및 2,500 ppm으로 나타났으며, Staphylococous epidermidis에 대한 MIC는 자미가 2,500 ppm으로 가장 낮은 농도를 나타내었다. 자색고구마 추출물의 화장품활성을 살펴본 결과 미백활성은 자미 품종 물 추출물에서 60% 이상으로 가장 높았으며, 자미 알콜추출물의 경우도 48.7%의 높은 미백활성을 나타내었다. 모든 자색고구마 품종에서 수렴활성과 주름개선효과는 관찰할 수 없었으나, 항염증 활성은 높게 관찰되었으며, 자미 품종의 에탄올 추출물이 94.4%로 가장 높게 나타났다. 자미 추출물을 첨가한 화장품의 온도 및 자연광에 의한 성상 변화는 확인되지 않았으며, 첩포시험을 통한 자각 증상도 나타나지 않았다. 또한 크림 사용 후 피부톤의 변화와 모공의 크기 변화를 측정한 결과 노화가 많이 일어난 피부가 젊은 피부보다 더 높은 긍정적인 효과를 나타내었다.

• Nutrition Research and Practice
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• 제15권6호
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• pp.798-806
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• 2021

BACKGROUND/OBJECTIVES: Obesity is associated with chronic inflammation. The spleen is the largest organ of the lymphatic system and has an important role in immunity. Obesity-induced inflammatory responses are triggered by Toll-like receptor (TLR)-myeloid differentiation primary response 88 (MyD88) pathway signaling. Phenethyl isothiocyanate (PEITC) and 3,3'-diindolylmethane (DIM), major dietary glucosinolates present in cruciferous vegetables, have been reported to produce anti-inflammatory effects on various diseases. However, the effects of PEITC and DIM on the obesity-induced inflammatory response in the spleen are unclear. The purpose of this study was to examine the anti-inflammatory effects of PEITC and DIM on the spleen and their mechanism in high fat/cholesterol diet (HFCD)-fed C57BL/6 mice. MATERIALS/METHODS: We established an animal model of HFCD-induced obesity using C57BL/6 mice. The mice were divided into six groups: normal diet with AIN-93G diet (CON), high fat diet (60% calories from fat) with 1% cholesterol (HFCD), HFCD with PEITC 30 mg/kg/day or 75 mg/kg/day (HFCD+P30, HFCD+P75), and HFCD with DIM 1.5 mg/kg/day or 7.5 mg/kg/day (HFCD+D1.5, HFCD+D7.5). Enzyme-linked immunosorbent assay was used to evaluate pro-inflammatory cytokine secretion. Western blot and quantitative polymerase chain reaction were used to analyze protein and mRNA levels of nuclear factor kappa B (NF-κB) p65, interleukin 6 (IL-6), cyclooxygenase 2 (COX-2), TLR2, TLR4, and MyD88 in spleen tissue. RESULTS: Serum IL-6 levels were significantly higher in the HFCD group than in groups fed a HFCD with PEITC or DIM. Levels of NF-κB p65 protein and TLR2/4, MyD88, NF-κB p65, IL-6, and COX-2 mRNA were significantly higher in the HFCD group than in the CON group and were reduced by the PEITC and DIM supplements. CONCLUSIONS: PEITC- and DIM-supplemented diets improved splenic inflammation by modulating the TLR2/4-MyD88 pathway in HFCD-fed mice. We suggest that dietary glucosinolates may at least partially improve obesity-induced inflammation of the spleen.

• Journal of Applied Biological Chemistry
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• 제64권3호
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• pp.197-203
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• 2021

본 연구에서는 보리 어린 순의 기능성 성분인 saponarin의 정량적 추출을 위한 최적의 추출 분석 조건을 methanol, ethanol, acetonitrile 및 water를 대상으로 반응표면분석법을 통해 실시하였다. 이를 통해 용매의 농도, 추출 시간에 따른 saponarin과 색소 추출율을 확인한 결과, 53.7% methanol 수용액에서 3.9 h 동안 진탕추출하는 것이 높은 saponarin 추출율을 유지하면서, 불필요한 색소의 추출을 최소화하는 최적 추출조건으로 확인되었다. 보리 어린 순의 재배에 필요한 인공광의 saponarin 생성 영향 평가를 광 주기, 광량, 광질을 달리한 조건에서 시험한 결과, saponarin 고함유 보리 어린 순의 인공광 최적 재배 조건은 8 h day^-1의 광 주기에서 6500K LED와 적색보광을 통한 총 광량 220-320 μmol m^-2 s^-1 에서 가장 효과적임을 확인 할 수 있었고, 청색광이 saponarin 생합성에 주요 인자로 작용함을 확인할 수 있었다.

• Korean Journal of Acupuncture
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• 제38권3호
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• pp.140-150
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• 2021

Objectives : The increase of osteoclasts could cause osteoporosis and bone-related diseases. Also, the inhibition of osteoclast differentiation is important in treating bone-related diseases. Traditionally, Psoraleae Semen has been used for geriatric diseases, aging and musculoskeletal diseases. The purpose of this study is to investigate the effect of Psoraleae Semen ethanol extract (PS) on osteoclast differentiation and its function. Methods : To confirm the effect of PS on osteoclastogenesis and bone resorption activity, various levels of concentrations of PS (5, 10, 20 and 40 ㎍/ml) were tested on RAW 264.7 cells cultured with RANKL. We measured tartarate-resistant acid phosphatase (TRAP)-positive cells, TRAP activity, pit formation and F-actin ring formation. The expressions of nuclear factor of activated T-cells (NFATc1) and c-Fos were confirmed through western blot and reverse transcription- polymerase chain reaction (RT-PCR). Also, the expression of bone resorption and fusion-related genes in osteoclast was confirmed by RT-PCR. Results : PS decreased the number of TRAP-positive cells and the TRAP activity. In addition, PS significantly inhibited the formation of pit and F-actin ring. Furthermore, PS decreased the expression of osteoclast related genes. Conclusions : PS inhibits osteoclast differentiation and bone resorption ability through inhibition of the expression of osteoclast-related genes. This indicates that PS may be a potential therapeutic agent to osteoporosis by suppressing osteoclastogenesis.

• Animal Bioscience
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• 제34권10호
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• pp.1590-1599
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• 2021

Objective: This study investigates the expression patterns of toll-like receptors (TLRs) and intracellular mediators in horse muscle cells after exercise, and the relationship between TLRS expression in stressed horse muscle cells and immune cell migration toward them. Methods: The expression patterns of the TLRs (TLR2, TLR4, and TLR8) and downstream signaling pathway-related genes (myeloid differentiation primary response 88 [MYD88]; activating transcription factor 3 [ATF3]) are examined in horse tissues, and horse peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells (PMNs) and muscles in response to exercise, using the quantitative reverse transcription-polymerase chain reaction (qPCR). Expressions of chemokine receptor genes, i.e., C-X-C motif chemokine receptor 2 (CXCR2) and C-C motif chemokine receptor 5 (CCR5), are studied in PBMCs and PMNs. A horse muscle cell line is developed by transfecting SV-T antigen into fetal muscle cells, followed by examination of muscle-specific genes. Horse muscle cells are treated with stressors, i.e., cortisol, hydrogen peroxide (H₂O₂), and heat, to mimic stress conditions in vitro, and the expression of TLR4 and TLR8 are examined in stressed muscle cells, in addition to migration activity of PBMCs toward stressed muscle cells. Results: The qPCR revealed that TLR4 message was expressed in cerebrum, cerebellum, thymus, lung, liver, kidney, and muscle, whereas TLR8 expressed in thymus, lung, and kidney, while TLR2 expressed in thymus, lung, and kidney. Expressions of TLRs, i.e., TLR4 and TLR8, and mediators, i.e., MYD88 and ATF3, were upregulated in muscle, PBMCs and PMNs in response to exercise. Expressions of CXCR2 and CCR5 were also upregulated in PBMCs and PMNs after exercise. In the muscle cell line, TLR4 and TLR8 expressions were upregulated when cells were treated with stressors such as cortisol, H₂O₂, and heat. Migration of PBMCs toward stressed muscle cells was increased by exercise and oxidative stresses, and combinations of these. Treatment with methylsulfonylmethane (MSM), an antioxidant on stressed muscle cells, reduced migration of PBMCs toward stressed muscle cells. Conclusion: In this study, we have successfully cultured horse skeletal muscle cells, isolated horse PBMCs, and established an in vitro system for studying stress-related gene expressions and function. Expression of TLR4, TLR8, CXCR2, and CCR5 in horse muscle cells was higher in response to stressors such as cortisol, H₂O₂, and heat, or combinations of these. In addition, migration of PBMCs toward muscle cells was increased when muscle cells were under stress, but inhibition of reactive oxygen species by MSM modulated migratory activity of PBMCs to stressed muscle cells. Further study is necessary to investigate the biological function(s) of the TLR gene family in horse muscle cells.

• 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.496-501
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• 2009

저장조건을 달리한 딸기 화분을 FCR Test와 결실률을 바탕으로 하여 화분 활력을 조사하고 암술 주두 활력을 인공수분을 통하여 구명함으로서 딸기 교배 육종시 효율성을 높이고자 하였다. '설향'을 대상으로 화분 활력을 조사하였으며 암술 주두의 수정 능력은 '매향' 등 4품종을 공시하고 화분친은 '설향'을 사용하였다. 딸기 화분은 상대습도 33%이하의 건조한 조건에서는 처리 온도($18^{\circ}C$ 및 $22^{\circ}C$)와 관계없이 수일 동안 높은 활력을 유지하였다. 반면 상대습도가 높을수록 화분 활력의 감소가 컸으며 저장한지 7일차에 대부분의 화분이 활력을 상실하여 결실률이 급격히 감소하였고 이러한 경향은 $18^{\circ}C$보다 $22^{\circ}C$저장 조건에서 결실률이 더욱 불량하였다. 즉, 상대습도가 낮은 경우 화분 활력은 크게 감소하지 않았으며, 온도보다는 습도가 화분 활력을 결정하는 중요한 요인으로 작용하는 것으로 판단된다. 암술의 수정 능력은 품종간의 차이가 있었으나 시설 내부 일평균온도가 $15^{\circ}C$내외일때, 제웅 후 2일차부터 8일차까지 평균 77.2%이상으로 높게 유지되었다. 그러나 결실률은 제웅 후 10일차부터 점차 감소하기 시작하여 12일차 이후에는 일부 품종을 제외하고 급격히 감소하였다. 적산온도를 기준으로 하였을 때, 제웅 후 $45{\sim}140^{\circ}C$내외 범위에서 인공수분을 하는 것이 딸기 결실률을 향상 시킬 수 있을 것으로 판단된다.