• 대한본초학회지
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• 제24권4호
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• pp.215-224
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• 2009

Objectives : Atopic dermatitis is a chronic inflammatory disease characterized by typically distributed eczematous skin lesion with pruritus, lichenification and dry skin. In this study, we performed to assess the therapeutic effects of co-treatment of Chenilyeomgamibang (CGB) and Chenggihaedok-san (CHS, C&C) on the TNCB(trinitrochlorobenzene)-induced atopic dermatitis in NC/Nga mice, characterized by the onset of atopic dermatitis along with an increase the number of inflammatory cells and dysregulation of Th2 cytokines. Methods : Defined amount of CGB was sprayed on mice skin and CHS was simultaneously orally administrated to TNCB treated NC/Nga mice for 5 weeks. The immune cell types were caracterized by flow cytometry using each specific antibody. The amount of Th2 cytokines in serum and splenocytes culture supernatant were measured by ELISA. Results : Administration of C&C significantly reduced clinical dermatitis severity including pruritus, edema, eczematous and erythema. Histological findings indicated that the thickening of epidermis/dermis and dermal infiltration of inflammatory cells were dramatically reduced. Flow cytometry analysis showed that infiltrated immune cell numbers of CCR3+, B220+/IgE+, Gr-1+/CD11b+, and CD117+ were significantly reduced in C&C-treated dorsal skin lesion. Furthermore, T cell composition rate in PBMC was also dramatically decreased by the treatment. C&C greatly down-regulated production of Th2 cytokines including IL-4, IL-5, IL-13 in the serum. The down- regulatory effects of C&C on these Th2 cytokines production were also detected in CD3/ CD28 activated splenocytes. Conclusions : These results indicated that C&C is a plausible therapeutic agent for treatment of atopic dermatitis through regulating the Th2 skewed immune system.

• 한국가금학회지
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• 제36권2호
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• pp.177-186
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• 2009

형질전환 동물에 있어서 외래 유전자의 조절되지 않은 과다 발현은 생리적인 부작용이나 독성을 나타내게 된다. 본 연구에서는 이러한 문제를 해결하기 위하여 외래 유전자 발현 조절 system인 tetracycline-inducible expression system(Tet system)을 도입하였다. 그러나 종래의 Tet system을 이용한 유전자 발현 조절은 system 자체의 구성 요소로 인한 미미한 leaky 현상 때문에 완벽하게 이루어지지 않는다. 본 연구에서는 보다 완벽한 외래 유전자의 발현 조절 system을 구축하기 위하여 rtTA 대신 진일보한 형태의 $rtTA2^SM2$를 도입하고 TRE 부분을 TRE-tight로 대체하였다. 확립된 retrovirus vector system을 이용하여 다양한 표적세포와 형질전환 닭으로부터 혈소판 생산의 일차적인 조절자이며, 조혈간세포의 생존과 증식에 있어서 매우 중요한 역할을 하는 human thrombopoietin(hTPO)를 생산하고자 하였다. In vitro 상의 연구에서, CEF 세포에서 발현되는 hTPO가 가장 높은 발현량과 발현 유도율을 나타내었으며, 상업적으로 판매되고 있는 hTPO나 다른 표적세포에서 생산된 hTPO에 비해 생물학적 활성이 가장 높은 것으로 확인되었다. 고농도로 농축한 재조합 retrovirus를 stage X단계의 계란의 배반엽 층에 미세주입하여 대리 난각 방법으로 배양한 결과, 미세주입한 138개의 계란 중 21일 후에 15개의 계란에서 병아리가 부화하였으며, 그 중 8마리가 형질전환 개체로 확인되었다. 이 형질전환 닭은 사료 1 g 당 0.5 mg의 doxycycline을 첨가하여 2주간 식이하였으며, 그 후 혈액을 채취하여 hTPO 농도를 측정한 결과 200ng/mL로 확인되었다. 또한 형질전환 개체 중 수컷의 정자에서 hTPO 유전자의 존재를 확인함으로써 germLine transmission의 가능성을 입증하였다. 이상의 연구 결과는 사람의 cytokine 단백질의 대량생산을 위한 생체반응기로서의 형질전환 닭의 생산 가능성을 제시한 데 의의가 있다.

• 한국식품영양과학회지
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• 제43권11호
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• pp.1688-1694
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• 2014

본 연구는 한라봉 과피 추출물과 효소 처리시킨 한라봉 과피추출물의 3T3-L1 지방전구세포에서의 항비만 효과를 비교하여 연구하였다. Cytolase를 이용한 플라보노이드 형태 전환으로 한라봉 과피 추출물에 다량 존재하던 narirutin이 naringenin으로 또는 hesperidin이 hesperetin으로 변환되었다. 지방전구세포에 분화배지와 함께 10일간 HLB와 HLB-C, 양성대조군으로 Sinetrol을 다양한 농도로 처리한 결과 모든 세포군에서 0.5 mg/mL 농도에서는 세포생존율에 유의적인 영향을 미치지 않았기 때문에 적정 처리 농도를 0.5 mg/mL로 확정하였다. Oil red O 염색을 통해 지방세포의 분화 정도를 측정한 결과 양성대조군인 Sinetrol 처리군 보다 같은 농도의 HLB와 HLB-C 처리군이 분화를 억제시켰다. 지방세포 분화 억제의 분자생화학적 기전을 규명하기 위해 분화 관련 유전자와 단백질 발현을 RT- PCR과 western blotting을 통해서 실험한 결과, 0.5 mg/mL 농도의 HLB와 HLB-C 처리군에서 대조군과 Sinetrol군에 비해 $C/EBP{\alpha}$, SREBP1c의 유전자가 감소하는 경향성을 나타내었고 $C/EBP{\alpha}$와 $PPAR{\gamma}$의 단백질 발현이 유의적으로 감소하는 것을 확인하였다. 이미 형성된 지방세포의 분해 작용에 HLB와 HLB-C가 영향을 주는지 평가하기 위해 완전히 분화된 지방세포에 24시간 동안 0.5 mg/mL의 농도로 처리한 결과 Sinetrol과 HLB는 지방분해 효과를 나타내었으나 HLB-C는 지방세포의 분해 작용에 유의적인 영향을 주지 않았다. 따라서 이러한 실험 결과들은 한라봉 과피 추출물이 지방세포 분화 억제와 지질분해 활성이 있고 이는 cytolase로 효소 처리를 할 경우에도 비슷한 결과를 보였으며 오히려 지질분해 활성은 감소하였다. 한라봉 과피 추출물의 in vitro 상에서 검증된 항비만 효능이 향후에 in vivo 상에서 지방분화 억제와 지방분해에 미치는 영향에 대한 추가 검증이 필요하다고 사료된다. 또한 향후 한라봉뿐 아니라 다른 감귤류를 이용하여 다양한 probiotics 미생물에 존재하는 효소와 다른 상업화 효소 등을 통한 물질전환 효과를 비교 연구하는 것이 필요하다고 사료된다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.27-27
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• 2003

A diversified and concentrative approach of methylation player can be one of the most powerful studies in the understanding of global epigenetic modifications. Previous studies have suggested that DNA methylation contributes to transcriptional silencing through the several DNA methylation-mediated repression systems by hypermethylation, including methyltransferases (DNMTs), DNA methyltransferase association protein 1 (DMAPl), methyl-CpG binding domain (MBD), and histone deacetylases (HDACs). Assembly of these regulatory protein complexes act sequentially, reciprocally, and interdependently on the newly composed DNA strand through S phase. Therefore, these protein complexes have a role in coupling DNA replication to the designed turn-off system in genome. In this study, we attempted to address the role of DNA methylation by the functional analysis of the methyltransferase molecule, we described the involvement of DMAP1 and DNMTs in cell divistion and the effect of their loss. We also described distinct patterns that DMAP1 and DNMTs are spatially reorganized and displaced from condensing chromosomes as cells progress through mitosis in HeLa cell, COS7, and HIH3T3 cell cycle progressions. DNMT1, DNMT3b, and DMAP1 do not stably contact the genetic material during chromosome compaction and repressive expression. These finding show that the loss of activities of DNMTs and DMAP1 occure stage specifically during the cell cycle, may contribute to the integral balance of global DNA methylation. This is consistent with previous studies resulted in decreased histone acetyltransferases and HDACs, and differs from studies resulted in increased histone methyltransferases. Our results suggest that DNA methylation by DNMTs and DMAP1 during mitosis acts to antagonize hypermethylation by which this mark is epigenetical mitotic-specific methylation.

• Tuberculosis and Respiratory Diseases
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• 제70권3호
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• pp.206-217
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• 2011

Background: Transcription factor FOXP3 characterizes the thymically derived regulatory T cells. FOXP3 is expressed by cancer cell itself and FOXP3 expression was induced by TGF-${\beta}$ treatment in pancreatic cancer cell line. However, the expression of FOXP3 expression is not well known in patients with lung cancer. This study was conducted to investigate the expression of FOXP3 in patients with lung cancer and to investigate the regulation of FOXP3 expression by the treatment of TGF-${\beta}$ and DNA methyltransferase inhibitor in lung cancer cell lines. Methods: FOXP3 expression in the tissue of patients with resected non-small cell lung cancer (NSCLC) was evaluated by immunohistochemistry. The regulation of FOXP3 expression was investigated by Western blot and RT-PCR after lung cancer cell lines were stimulated with TGF-${\beta}1$ and TGF-${\beta}2$. The regulation of FOXP3 expression was also investigated by RT-PCR and flow cytometry after lung cancer cell lines were treated with DNA methyltransferase inhibitor (5-AZA-dC). Results: FOXP3 expression was confirmed in 27% of patients with NSCLC. In NCI-H460 cell line, TGF-${\beta}2$ decreased FOXP3 mRNA and protein expressions. In A549 cell line, both TGF-${\beta}1$ and TGF-${\beta}2$ decreased FOXP3 mRNA and protein expressions. 5-AZA-dC increased FOXP3 mRNA expression in NCI-H460 and A549 cell lines. Moreover, 5-AZA-dC increased intracellular FOXP3 protein expression in A549 cell lines. Conclusion: It was shown that FOXP3 is expressed by cancer cell itself in patients with NSCLC. Treatment of TGF-${\beta}2$ and DNA methyltransferase inhibitor seems to be associated with the regulation of FOXP3 expression in lung cancer cell lines.

• Clinical and Experimental Pediatrics
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• 제56권11호
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• pp.482-489
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• 2013

Purpose: The aim of the present study was to investigate the differences in lower airway inflammatory immune responses, including cellular responses and responses in terms of inflammatory mediators in bronchoalveolar lavage fluid (BALF) and the airway, to rhinovirus (RV) infection on asthma exacerbation by comparing a control and a murine asthma model, with or without RV infection. Methods: BALB/c mice were intraperitoneally injected with a crude extract of Dermatophagoides farinae (Df ) or phosphate buffered saline (PBS) and were subsequently intranasally treated with a crude extract of Df or PBS. Airway responsiveness and cell infiltration, differential cell counts in BALF, and cytokine and chemokine concentrations in BALF were measured 24 hours after intranasal RV1B infection. Results: RV infection increased the enhanced pause (Penh) in both the Df sensitized and challenged mice (Df mice) and PBS-treated mice (PBS mice) (P<0.05). Airway eosinophil infiltration increased in Df mice after RV infection (P<0.05). The levels of interleukin (IL) 13, tumor necrosis factor alpha, and regulated on activation, normal T cells expressed and secreted (RANTES) increased in response to RV infection in Df mice, but not in PBS mice (P<0.05). The level of IL-10 significantly decreased following RV infection in Df mice (P<0.05). Conclusion: Our findings suggest that the augmented induction of proinflammatory cytokines, Th2 cytokines, and chemokines that mediate an eosinophil response and the decreased induction of regulatory cytokines after RV infection may be important manifestations leading to airway inflammation with eosinophil infiltration and changes in airway responsiveness in the asthma model.

• 생명과학회지
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• 제17권1호
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• pp.56-63
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• 2007

인슐린은 지방세포 또는 근육세포에서 포도당 흡수 조절 통로단백질이 함유되어 있는 소포제를 세포막으로의 이동을 촉진시킨다. 우리는 여기서 지방세포로의 분화는 인슐린에 의한 포도당 흡수에 대한 반응이 증가됨을 보였다. 반면에 지방세포로의 분화는 PDGF에 의한 포도당 흡수 반응이 감소됨을 보였다. 인슐린 수용체나 caveolae는 지방세포로의 분화과정 동안 발현이 증가된다. 또한 지방세포로의 분화는 인슐린에 의한 Akt의 활성을 증가시켰다. 하지만 PDGF에 의한 Akt의 활성은 크게 감소하였다. 하지만 인슐린은 지방세포 또는 섬유아 전구세포에서 ERK의 활성을 유도하지 않았다. PDGF에 의한 ERK 활성 또한 지방세포로의 분화과정에 따라 감소하였다. P13K의 저해제인 LY294002는 지방세포 뿐만 아니라 섬유아 전구세포에서 인슐린에 의한 포도당 흡수를 저해하였다. 마지막으로 인슐린 수용체, Akt, SHIP2, p85등이 lipid raft/caveolae에 존재함을 확인하였고 인슐린에 의해 이런 단백질들이 lipid raft/caveolae로 이동함을 관찰하였다. 이런 결과를 토대로 lipid raft는 포도당 홉수를 위한 인슐린의 기능적 작용을 하는데 매우 중요한 환경을 제공함을 주장한다.

• 한국수정란이식학회지
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• 제31권1호
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• pp.1-7
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• 2016

Xenotransplantation involves multiple steps of immune rejection. The present study was designed to produce nuclear transfer embryos, prior to the production of transgenic pigs, using fibroblasts carrying transgenes human complement regulatory protein hCD59 and interleukin-18 binding protein (hIL-18BP) to reduce hyperacute rejection (HAR) and cellular rejection in pig-to-human xenotransplantation. In addition to the hCD59-mediated reduction of HAR, hIL-18BP may prevent cellular rejection by inhibiting the activation of natural killer cells, activated T-cell proliferation, and induction of $IFN-{\gamma}$. Transgene construct including hCD59 and ILI-18BP was introduced into miniature pig fetal fibroblasts. After antibiotic selection of double transgenic fibroblasts, integration of the transgene was screened by PCR, and the transgene expression was confirmed by RT-PCR. Treatment of human serum did not affect the survival of double-transgenic fibroblasts, whereas the treatment significantly reduced the survival of non-transgenic fibroblasts (p<0.01), suggesting alleviation of HAR. Among 337 reconstituted oocytes produced by nuclear transfer using the double transgenic fibroblasts, 28 (15.3%) developed to the blastocyst stage. Analysis of individual embryos indicated that 53.6% (15/28) of embryos contained the transgene. The result of the present study demonstrates the resistance of hCD59 and IL-18BP double-transgenic fibroblasts against HAR, and the usefulness of the transgenic approach may be predicted by RT-PCR and cytolytic assessment prior to actual production of transgenic pigs. Further study on the transfer of these embryos to surrogates may produce transgenic clone miniature pigs expressing hCD59 and hIL-18BP for xenotransplantation.

• 식품과학과 산업
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• 제52권3호
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• pp.261-271
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• 2019

NGS 기술이 발전함에 따라 우리 몸의 생리와 면역조절에 있어서 장내미생물의 중요성이 알려지면서부터 장내미생물군집의 구조를 직접 조절할 수 있는 프로바이오틱스의 중요성 역시 재조명 받고 있다. 인류는 프로바이오틱스를 오랫동안 발효식품 등을 통하여 섭취하였는데, 프로바이오틱스는 식품의 보존성 및 영양성을 높일 뿐 아니라 인체의 건강에 이로운 역할을 한다. 특히 프로바이오틱스의 섭취는 생체 내에서 Treg의 기능을 활성화하여 장내 환경을 개선시켜 유익한 장내미생물의 생육을 도우며, 염증반응, 알러지질환, 자가면역질환 등을 완화시키는 효과가 있다. 특히 프로바이오틱스는 장내 유익균인 Bifidobacterium, Faecalibacterium, Akkermansia 및 Bacteroides 속 미생물의 빈도를 증가시키고, 이들은 단쇄지방산 및 신체에 이로운 대사체 등을 생산한다. 지금까지 프로바이오틱스는 대부분 건강기능식품으로 사용되어 왔으나, 최근 들어 장내 유익균에 대한 기능성이 알려지면서 기존 프로바이오틱스를 포함한 장내 미생물을 이용한 NGPs 개발이 활발히 진행되고 있다. 하지만 NGPs 개발에는 여전히 한계가 존재한다. 아직까지 장내 미생물의 분리, 동정은 일반 세균 배양에 비해 매우 까다롭고, 특별한 배양 기술이 필요하므로 현재까지 NGPs로 활용될 수 있는 장내 미생물은 매우 제한적이다. 또한 기존 프로바이오틱스와는 다르게 NGPs는 의약품처럼 전임상, 독성시험, 약물역학, 3단계의 임상시험을 거쳐야 한다. 하지만 기존 프로바이오틱스의 질병 개선 효과를 뛰어넘어 고형암, 대사질환 및 면역질환의 차세대 치료제로서의 활용 가능성이 매우 높기 때문에 앞으로 더 폭넓은 연구가 진행되어야 할 것이다.

• Journal of Animal Science and Technology
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• 제64권4호
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• pp.800-811
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• 2022

The integration of metabolomics and transcriptomics may elucidate the correlation between the genotypic and phenotypic patterns in organisms. In equine physiology, various metabolite levels vary during exercise, which may be correlated with a modified gene expression pattern of related genes. Integrated metabolomic and transcriptomic studies in horses have not been conducted to date. The objective of this study was to detect the effect of moderate exercise on the metabolomic and transcriptomic levels in horses. In this study, using nuclear magnetic resonance (NMR) spectroscopy, we analyzed the concentrations of metabolites in muscle and plasma; we also determined the gene expression patterns of branched chain (alpha) keto acid dehydrogenase kinase complex (BCKDK), which encodes the key regulatory enzymes in branched-chain amino acid (BCAA) catabolism, in two breeds of horses, Thoroughbred and Jeju, at different time intervals. The concentrations of metabolites in muscle and plasma were measured by ¹H NMR (nuclear magnetic resonance) spectroscopy, and the relative metabolite levels before and after exercise in the two samples were compared. Subsequently, multivariate data analysis based on the metabolic profiles was performed using orthogonal partial least square discriminant analysis (OPLS-DA), and variable important plots and t-test were used for basic statistical analysis. The stress-induced expression patterns of BCKDK genes in horse muscle-derived cells were examined using quantitative reverse transcription polymerase chain reaction (qPCR) to gain insight into the role of transcript in response to exercise stress. In this study, we found higher concentrations of aspartate, leucine, isoleucine, and lysine in the skeletal muscle of Jeju horses than in Thoroughbred horses. In plasma, compared with Jeju horses, Thoroughbred horses had higher levels of alanine and methionine before exercise; whereas post-exercise, lysine levels were increased. Gene expression analysis revealed a decreased expression level of BCKDK in the post-exercise period in Thoroughbred horses.