• 생명과학회지
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• 제29권10호
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• pp.1159-1163
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• 2019

남성호르몬 감소와 관련된 남성갱년기에 대한 관심이 고조되고 있지만, 남성호르몬의 정량을 위해 항체를 이용하는 고가의 kit가 이용되고 있다. 본 연구에서는 in vitro 전사 활성 시험법을 이용하여 남성 스테로이드호르몬의 활성 혹은 농도를 검증하는 시스템을 구축하였다. 테스토스테론-AR (androgen receptor) 복합체와 반응하는 ARE-AdE1bTATA 염기서열이 삽입되고 리포터로 luciferase를 발현하는 테스토스테론 유사활성 검증 리포터 플라스미드인 pGL2-Neo-ARE-AdE1BTATA를 제조하고, 인체 전립선암 세포인 LNcap-LN3 세포에 stable transfection을 실시하였다. 구축된 LNcap-LN3/pGL2-Neo-ARE-AdE1BTATA TCD (testosterone compound detection) 시스템은 표준물질인 테스토스테론의 $10^{-13}{\sim}10^{-8}M$ 범위에서 농도 증가에 비례하는 정량성을 보였다. 이 연구에서 확립된 in vitro TCD 시스템을 이용하면 천연물 유래 테스토스테론 유사물질 및 테스토스테론 저하물질의 대량 탐색 등이 가능할 것이므로, 건강기능성 식품이나 의약품 신소재의 개발에 기여할 것이다.

• 동의생리병리학회지
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• 제18권3호
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• pp.740-746
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• 2004

In order to identify the anti-inflammatory and analgesic properties of natural herbal extracts, widely used in the Korean traditional medicine, an in vitro screening system was designed using pGL3, a luciferase reporter vector, and the tumor necrosis factor (TNF)-α and cyclooxygenase (COX)-II as target genes. The promoter regions of each gene was generated by PCR using the human chromosome as template DNA, and inserted into pGL3 vector with Kpnl and Hindlll. The final construct was transfected into human myleomonocytic leukemia cells (U937) that could be differentiated and activated by phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS). Using this system, we tested the anti-inflammatory and analgesic effects of several herbal extracts being regarded to have the medicinal effects of diminishing the body heat and complementing Qi. The well-known chemicals of PD98059 and berberine chloride were used as controls of the transcriptional inhibitors of TNF-α and COX-II, respectively. Among them, Salvia miltiorrhiza (Dan-Sam) was found to exhibit the significant medicinal properties of anti-inflammatory and analgesic effects.

• 한국발생생물학회지:발생과생식
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• 제14권2호
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• pp.123-129
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• 2010

배아 줄기세포는 미분화상태에서 자가 재생을 유지할 수 있다. 자가 재생은 OCT4, SOX2와 NANOG와 같은 많은 인자들이 작용한다. 생쥐 배아 줄기세포에서 OCT4와 SOX2가 Nanog 프로모터에 결합하여 Nanog 유전자의 발현을 촉진한다는 사실은 생쥐 promoter에 관한 정밀분석으로 알려져 있다. 본 연구에서는 인간 Nanog promoter를 정밀 분석하기 위해 연속적인 결손 돌연변이를 가진 promoter-reporter construct를 제조하였다. Promoter의 최대 활성은 0.6 kb(-253/+365) promoter-reporter construct에서 발견되었으며, 이 construct에는 OCT4 및 SOX2의 결합부위가 포함된다. OCT4와 SOX2의 기여도를 확인하기 위하여 OCT4 및 SOX2의 결합부위에 자리 특이적 돌연변이를 유도하고 promoter 활성에 미치는 영향을 조사한 결과, OCT4나 SOX2 어느 한 군데라도 돌연변이가 존재하면 promoter 활성이 현저히 저해되었다. 본 연구 결과를 통해 인간 Nanog 유전자 발현에 있어 OCT4 및 SOX2가 필수적임을 직접적으로 확인할 수 있었다.

• Reproductive and Developmental Biology
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• 제30권2호
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• pp.81-85
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• 2006

This study was conducted to examine the effect of IRES controlled reporter gene on screening and production of recombinant human erythropoietin (EPO) proteins from cultured CHO cells. The cDNA was cloned for EPO from human liver cDNA Using site-directed mutagenesis, we generated recombinant human EPO (rhEPO) with two additional N-glycosylations (Novel erythropoiesis-stimulating protein: NESP). Wild type hEPO and NESP were cloned into expression vectors with GFP reporter gene under regulatory control of CMV promoter and IRES so that the vectors could express both rhEPO and GFP. The expression vectors were transfected to cultured CHO-K1 cells. Under microscopy, expression of GFP was visible. Using supernatant of the culture, ELISA assay, immunocytochemistry and in vitro assay using EPO dependant cell line were performed to estimate biological activity to compare the production characteristics (secretion levels, etc.) between rhEPO and NESP. The activity of NESP protein, obtained by mutagenesis, was described and compared with its rhEPO counterpart produced under same conditions. Although NESP had less secretion level in CHO cell line, the biological activity of NESP was greater than that of rhEPO. These results are consistent with previous researches. We also demonstrated that rhEPO and GFP proteins expressed simultaneously from transfected CHO cell line. Therefore we conclude that use of GFP reporter gene under IRES control could be used to screen and produce rhEPO in cultured CHO cells.

• 한국언론정보학보
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• 제26권
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• pp.45-82
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• 2004

본 연구는 인터넷 신문이 시민기자 제도의 도입을 통해 시민 저널리즘을 보다 적극적으로 구현할 수 있을 것이라는 전제하에 시민기자 제도를 도입하고 있는 인터넷 신문과 그렇지 않은 인터넷 신문간에 시민 저널리즘 실천 정도에 있어 유의미한 차이가 있는지를 분석했다. 또 시민기자 제도를 도입하고 있는 인터넷 신문을 시민 저널리즘 유형에 따라 기자 주도형 시인 저널리즘, 기자 시민 공동 주도형 시민 저널리즘, 시민 주도형 시민 저널리즘 등 3개 모델로 유형화한 뒤 각각의 시민 저널리즘 모델간에 시인 저널리즘 실천 정도에 있어 유의미한 차이가 있는지를 분석했다. 이를 위해 10개 항목으로 구성된 시민 저널리즘 척도를 사용해 시민 저널리즘 실천 정도를 분석한 결과 시인기자 제도를 도입한 인터넷 신문의 시민 저널리즘 지수가 시인기자 제도를 도입하지 않은 인터넷 신문보다 높게 나타났다. 또 기자 시민 공동 주도형 시민 저널리즘 모델이 시민 주도형 시민 저널리즘 모델보다 시민 저널리즘 지수가 높은 것으로 분석됐다. 그러나 시민기자 제도를 도입하고 있는 인터넷 신문 기사의 평균 시민 저널리즘 지수는 <오마이뉴스> 2.55, <시민의신문> 2.47, <넷피니언> 1.59 등으로 세 신문모두 시민 저널리즘 실천 정도가 다소 낮은 것으로 나타났다. 요컨대 시민 기자 계도를 도입한 인터넷 신문이 그렇지 않은 신문에 비해 시민 저널리즘 지수가 높게 나타나고 또 시민 저널리즘 유형에 따라 시민 저널리즘 실천 정도에 있어 차이가 있기는 하지만 전반적으로 시민기지 제도를 도입하고 있는 한국 인터넷 신문의 일상적인 시민 저널리즘 실천 정도는 그다지 높지 않은 것으로 분석됐다.

• 한국응급구조학회지
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• 제11권2호
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• pp.51-66
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• 2007

Purpose: With a view to providing basic data to develop cardiopulmonary resuscitation education suitable for elementary students, the cardiopulmonary resuscitation education was conducted to grasp students' knowledge, skills accuracy and the attitude change before and after the education. Methods: Convenience sampling was made on fourth and fifth graders(total-35 students) of S elementary school located in K city, Chungcheongnam-do, and this was a pre-experiment research designed before and after choosing a single group. In terms of methods, specifically we, researchers ; 1) Handed out questionnaires to students directly to make them fill in firsthand and collected the questionnaires. 2) Utilized PPT materials based on 2005 AHA guideline and DVD materials of AHA, to give students theoretical education of cardiopulmonary resuscitation. We used Anne/SkillReporter$^{(R)}$ torso produced by Leardal Inc, and Little Anne to conduct practical education individually. 3) Asked students to give Anne/SkillReporter$^{(R)}$ torso cardiopulmonary resuscitation five times with the ratio of 30 : 2, and then one of researchers filled in the evaluation sheet individually. 4) Evaluated the accuracy of students' ability to perform the resuscitation based on the record of Anne/SkillReporter$^{(R)}$ integrated printer(which was the objective tool to grasp students' skills accuracy). 5) Gave out questionnaires to make students fill them in and then collected them. after completing the practical evaluation. Results: 1) In case of the attitude about cardiopulmonary resuscitation, Students' confidency rose from 19.28%(before the education) to 93.57(after the education)- which is a positive change. 2) As the result of the education, some elementary students scored 11 points (full score-16 points), up from 5 points before the education, in terms of the knowledge about cardiopulmonary resuscitation. The average point also reached 13.14 points(after the education), jump from 8.37(before the education), which was the rise of 29.8%. 3) When it comes to the practical performance, the skills accuracy was 80.93% on average, and the calculation method was as follows: total items were 16, and each item was marked form 0 to 2 points, meaning the full score was 32 points. The minimum score was 19 points and the maximum was 32($M{\pm}SD=25.90{\pm}2.88$), which was calculated based on percentage. 4) Regarding skills accuracy, respiration accuracy(%)($M{\pm}SD=30.20{\pm}27.16$) was higher than pressure accuracy(%) ($M{\pm}SD=15.34{\pm}25.27$). Conclusion: The result showed that students' attitude on cardiopulmonary resuscitation changed positively. and meaningful difference(p = .00) existed in the change of students' knowledge. In terms of skills accuracy. chest compression and airway control showed high accuracy, but the result of Anne/SkillReporter$^{(R)}$ performance showed that the accuracy of chest compression was lower than that of mouth-to-mouth resuscitation.

• Journal of Periodontal and Implant Science
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• 제36권4호
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• pp.839-847
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• 2006

The aim of this study is to monitor reporter gene expression under osteocalcin gene promoter, using a real-time molecular imaging system, as tool to investigate osteoblast differentiation. The promoter region of mouse osteocalcin gene 2 (mOG2), the best-characterized osteoblast-specific gene, was inserted in promoterless luciferase reporter vector. Expression of reporter gene was confirmed and relationship between the reporter gene expression and osteoblastic differentiation was evaluated. Gene expression according to osteoblstic differentiation on biomaterials, utilizing a real-time molecular imaging system, was monitored. Luciferase was expressed at the only cells transduced with pGL4/mOGP and the level of expression was statistically higher at cells cultured in mineralization medium than cells in growth medium. CCCD camera detected the luciferase expression and was visible differentiation-dependent intensity of luminescence. The cells produced osteocalcin with time-dependent increment in BMP-2 treated cells and there was difference between BMP-2 treated cells and untreated cells at 14days. There was difference at the level of luciferase expression under pGL4/mOGP between BMP-2 treated cells and untreated cells at 3days. CCCD camera detected the luciferase expression at cells transduced with pGL4/mOGP on Ti disc and was visible differentiation-dependent intensity of luminescence This study shows that 1) expression of luciferase is regulated by the mouse OC promoter, 2) the CCCD detection system is a reliable quantitative gene detection tool for the osteoblast differentiation, 3) the dynamics of mouse OC promoter regulation during osteoblast differentiation is achieved in real time and quantitatively on biomaterial. The present system is a very reliable system for monitoring of osteoblast differentiation in real time and may be used for monitoring the effects of growth factors, drug, cytokines and biomaterials on osteoblast differentiation in animal.

• Molecules and Cells
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• 제41권5호
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• pp.423-435
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• 2018

This investigation was aimed at working out the combined role of lncRNA H19, miR-29b and Wnt signaling in the development of colorectal cancer (CRC). In the aggregate, 185 CRC tissues and corresponding para-carcinoma tissues were gathered. The human CRC cell lines (i.e. HT29, HCT116, SW480 and SW620) and normal colorectal mucosa cell line (NCM460) were also purchased. Si-H19, si-NC, miR-29b-3p mimics, miR-29b-3p inhibitor, si-PGRN and negative control (NC) were, respectively, transfected into the CRC cells. Luciferase reporter plasmids were prepared to evaluate the transduction activity of $Wnt/{\beta}-catenin$ signaling pathway, and dual-luciferase reporter gene assay was arranged to confirm the targeted relationship between H19 and miR-29b-3p, as well as between miR-29b-3p and PGRN. Finally, the proliferative and invasive capacities of CRC cells were appraised through transwell, MTT and scratch assays. As a result, overexpressed H19 and down-expressed miR-29b-3p displayed close associations with the CRC patients' poor prognosis (P < 0.05). Besides, transfection with si-H19, miR-29b-3p mimic or si-PGRN were correlated with elevated E-cadherin expression, decreased snail and vimentin expressions, as well as less-motivated cell proliferation and cell metastasis (P < 0.05). Moreover, H19 was verified to directly target miR-29b-3p based on the luciferase reporter gene assay (P < 0.05), and miR-29b-3p also bound to PGRN in a direct manner (P < 0.05). Finally, addition of LiCl ($Wnt/{\beta}-catenin$ pathway activator) or XAV93920 ($Wnt/{\beta}-catenin$ pathway inhibitor) would cause remarkably altered E-cadherin, c-Myc, vimentin and snail expressions, as well as significantly changed transcriptional activity of ${\beta}-catenin/Tcf$ reporter plasmid (P < 0.05). In conclusion, the lncRNA H19/miR-29b-3p/PGRN/Wnt axis counted a great deal for seeking appropriate diagnostic biomarkers and treatment targets for CRC.

• 한국응급구조학회지
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• 제12권1호
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• pp.17-26
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• 2008

Purpose : With a view to providing basic data to develop basic cardiopulmonary resuscitation education suitable for female college students, the basic cardiopulmonary resuscitation education was conducted to grasp students' knowledge, skill accuracy and the attitude change before and after the education. Methods : Convenience sampling was made on first graders(total-38 students) of K college located in K city, Chungcheongnam-do, and this was a preliminary research designed before and after choosing a single group. In terms of methods, the researchers as specified ; 1) Handed out questionnaires to students directly to make them fill in firsthand and collected the questionnaires. 2) Utilized Power Point materials based on 2005 AHA guideline and used Anne/Skill Reporter$^{(R)}$ torso produced by Leardal Inc, and Little Anne$^{(R)}$ to conduct practical education individually. 3) Asked students to give Anne/Skill Reporter$^{(R)}$ torso basic cardiopulmonary resuscitation five times with the ratio of 30:2, and then one of researchers filled in the evaluation sheet individually. 4) Evaluated the accuracy of students' ability to perform the resuscitation based on the record of Anne/Skill Reporter$^{(R)}$ integrated printer(which was the objective tool to grasp students' skills accuracy). 5) Gave out questionnaires to make students fill them in and then collected them, after completing the practical evaluation. Results : 1) In case of the attitude(confidence) about basic cardiopulmonary resuscitation, students' confidency rose from 14%(before the education) to 55.5%(after the education)- which was a positive change. 2) In case of the attitude(educational necessity) about basic cardiopulmonary resuscitation, students' confidency rose from 94.7%(before the education) to 100%(after the education)- which was a positive change. 3) As a result of the education, some female college students' scored knowledge about basic cardiopulmonary resuscitation. The average point also reached 88.95%(after the education), jump from 63.51%(before the education), which was the rise of 25.44%. 4) Regarding skill accuracy, pressure accuracy(%)($M{\pm}SD=91.37{\pm}14.16$) was higher than respiration accuracy(%)($M{\pm}SD=61.55{\pm}26.13$). Conclusion : The result showed that students' attitude(confidence, on basic cardiopulmonary resuscitation changed positively, and meaningful difference(p= .000) existed in the change of students' knowledge. Anne/Skill Reporter$^{(R)}$ performance showed that the accuracy of mouth-to-mouth resuscitation was lower than that of chest compression.

• Korean Journal of Radiology
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• 제21권6호
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• pp.726-735
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• 2020

Objective: Recent innovations in biology are boosting gene and cell therapy, but monitoring the response to these treatments is difficult. The purpose of this study was to find an MRI-reporter gene that can be used to monitor gene or cell therapy and that can be delivered without a viral vector, as viral vector delivery methods can result in long-term complications. Materials and Methods: CMV promoter-human organic anion transporting polypeptide 1B3 (CMV-hOATP1B3) cDNA or CMV-blank DNA (control) was transfected into HEK293 cells using Lipofectamine. OATP1B3 expression was confirmed by western blotting and confocal microscopy. In vitro cell phantoms were made using transfected HEK293 cells cultured in various concentrations of gadoxetic acid for 24 hours, and images of the phantoms were made with a 9.4T micro-MRI. In vivo xenograft tumors were made by implanting HEK293 cells transfected with CMV-hOATP1B3 (n = 4) or CMV-blank (n = 4) in 8-week-old male nude mice, and MRI was performed before and after intravenous injection of gadoxetic acid (1.2 µL/g). Results: Western blot and confocal microscopy after immunofluorescence staining revealed that only CMV-hOATP1B3-transfected HEK293 cells produced abundant OATP1B3, which localized at the cell membrane. OATP1B3 expression levels remained high through the 25th subculture cycle, but decreased substantially by the 50th subculture cycle. MRI of cell phantoms showed that only the CMV-hOATP1B3-transfected cells produced a significant contrast enhancement effect. In vivo MRI of xenograft tumors revealed that only CMV-hOATP1B3-transfected HEK293 tumors demonstrated a T1 contrast effect, which lasted for at least 5 hours. Conclusion: The human endogenous OATP1B3 gene can be non-virally delivered into cells to induce transient OATP1B3 expression, leading to gadoxetic acid-mediated enhancement on MRI. These results indicate that hOATP1B3 can serve as an MRI-reporter gene while minimizing the risk of long-term complications.