DNA 복제 시점의 차이를 응용한 불활성화 X-염색체에 대한 학습 자료의 모델

문선하 ( Moon Sun-ha ),전병균 ( Jeon Byeong-gyun ) 경상대학교 교육연구원 2023 현대교육연구 Vol.35 No.1

XY 성 결정 체계에 의해 암수가 결정되는 포유류의 경우, 두 X-염색체를 가지는 암컷에서 두 X-염색체 중의 하나는 불활성화되고, 이에 따른 여러 가지 성과 관련된 유전 현상이 나타난다. 이러한 X-염색체의 불활성화 및 후성학적인 유전자의 발현 등에 대한 개념을 을 좀 더 쉽게 이해할 수 있는 학습 자료 및 모델이 고등학교 교과서에서 잘 제시되지 않고 있다. 이에 이 연구에서는 DNA 복제 시점의 차이를 이용한 BrDu 표지 방법으로 두 X-염색체의 활성 및 불활성 상태를 좀 더 쉽게 구별하고, 이를 고등학교 생명과학 교과서에 응용할 수 있는 수업 자료를 개발하고자 하였다. 암컷의 섬유아세포를 사용하여 세포 주기를 동기화시킨 다음, S기의 초기 단계에 BrDu 표지를 실시하여, 핵형분석을 통해 X-염색체를 관찰하였다. 이에 S기의 초기에 복제되는 활성 X-염색체와 후반기에 복제되는 불활성 X-염색체를 명확하게 구분할 수 있었다. 또한, 불활성된 X-염색체의 탈메틸화는 후반기에서 전반기 단계로 DNA 복제 시점의 변화가 일어나는 것을 관찰하였다. 이 연구의 결과를 바탕으로 고등학교 생명과학 분야에서 염색체, 유전체, DNA, 유전자의 관계를 이해하고, 후성유전학 및 X-염색체 불활성화 개념을 가시적으로 이해할 수 있는 자료에 응용할 수 있을 것으로 판단된다. The XY chromosome is a main sex-determination system in most mammals. One of the two X-chromosomes is randomly inactivated in the female with two X-chromosomes, and the various sex-linked genetic and epigenetic expression are accompanied by the inactivated X-chromosome. However, the effective teaching model and materials for inactivated X-chromosome that students easily understandable are not presented in the high school life science textbook. The present study is to develop teaching model for application of inactivated X-chromosome with DNA replication assay using BrDu labeling. The female bovine fibroblasts were synchronized at G1 stage of cell cycle and labeled with BrDu. The fibroblasts were again treated with colcemid for synchronization of metaphase. Following BrDu labeling, the status of activated and inactivated X-chromosome was easily distinguished on the karyotyping, and DNA demethylation with SAH treatment was induced to activated X-chromosome from inactivated X-chromosome by change of DNA replication timing. Taken together, the present results have showed that learning model for inactivated X-chromosome and epigenetics can be applied by DNA replication timing assay in the high school life science textbook.

Inhibition of cell growth by cellular differentiation into adipocyte-like cells in dexamethasone sensitive cancer cell lines

김해인,문선하,이원철,이현정,Sharath Belame Shivakumar,이성호,박봉욱,노규진,전병균 한국통합생물학회 2018 Animal cells and systems Vol.22 No.3

The stress responses in human body lead to secretion of cortisol hormone. The present study investigated the cellular responses on cell growth and cellular differentiation into adipocytes by exposure of synthetic stress hormone, dexamethasone (DEX) in various human cancer and normal cells. After prolonged exposure of cells with 1 μg/ml DEX for 2 weeks, population doubling time (PDT) was significantly (P < .05) increased by inhibited cell growth in A-549 and MCF-7 cancer cells, and was unchanged in MDA-MB-231 cancer cells, normal MRC-5 fibroblasts, umbilical cord matrix-derived mesenchymal stem cells (UCMSCs) and dental papilla tissuederived mesenchymal stem cells (DSCs). Whereas, PDT was significantly (P < .05) decreased in U87-MG cancer cells by increased cell growth. Glucose uptake was significantly (P < .05) increased in all the cancer cell lines compared to that in normal cell lines. Further, adiposomelike vesicles were noted in A-549 and MCF-7 cancer cells indicating retarded cell growth by DEX treatment, and the vesicles were stained with Oil-Red O solution. Further, the expression of adipocyte-specific genes such as glucose transporter type 4 (GLUT4), glucocorticoid receptors β (GRβ) and peroxisome proliferator-activated receptor γ (PPARγ) were significantly (P < .05) increased in A-549 and MCF-7 with lipid vesicles. The level of telomerase activity was found to be significantly (P < .05) downregulated in DEX-treated A-549 and MCF-7 cancer cells. Our results have clearly shown that DEX treatment induces inhibition of cell growth by differentiating into adipocyte-like cells in dexamethasone sensitive cancer cells.

Synergic Effect on Adipocytic Differentiation by a Combination of Thyroxine and Glucocorticoid in A549 Cells

김현지,문선하,이성호,전병균 한국생명과학회 2023 생명과학회지 Vol.33 No.9

The present study examined the rate of cell growth and differentiation potential into adipocytes in A549 lung adenocarcinoma cells exposed to each adipogenic medium containing glucose metabolism hormones, such as thyroxine (T4) thyroid hormone and glucocorticoid (GC) adrenal steroid hormone, as well as pioglitazone (PGZ), a PPARγ agonist. Following each adipogenic treatment for 2 weeks, the rate of cell growth was significantly (p<0.05) inhibited, and the level of telomerase activity was significantly (p<0.05) decreased in the PGZ-based adipogenic medium containing both T4 and GC hormone compared with those containing each T4 or GC hormone. Moreover, the adiposome-like vesicles were highly reacted with Oil-Red O staining solution, and the levels of transcripts expressed in the differentiating adipocytes for adipogenesis, including adinopectin, leptin, and resistin, were significantly (p<0.05) increased in the PGZ-based adipogenic medium containing both T4 and GC hormone compared with those of the adipogenic medium containing each T4 or GC hormone, implying that adipocytic differentiation has fully occurred in the A549 cancer cells. Based on present observations, the PGZ-based adipogenic medium containing both T4 and GC efficiently induces inhibition of cell growth and cellular differentiation into adipocytes in A549 cancer cells rather than in the adipogenic medium containing only T4 or GC hormone. Adipogenic treatment could provide potential probability in cancer chemotherapy.

Relationship Between Serum Biochemical Markers of Myocardial Fibrosis and Diastolic Function at Rest and With Exercise in Hypertrophic Cardiomyopathy

심지영,하종원,최의영,이현진,문선하,김진미,임세중,정남식 대한심장학회 2009 Korean Circulation Journal Vol.39 No.12

Background and Objectives: Recent studies have demonstrated the usefulness of biochemical markers of collagen turnover as markers of myocardial fibrosis in various diseases. In this study, we hypothesized that increased collagen markers in patients with hypertrophic cardiomyopathy (HCM) were correlated with diastolic function at rest and diastolic functional reserve during exercise. Subjects and Methods: Thirty-six patients with HCM and 21 controls with normal left ventricular thickness were studied. Mitral septal annular velocities and mitral inflow velocities were measured at rest and during graded supine bicycle exercise (25 W, 3-minute increments) for the assessment of diastolic function at rest and during exercise. By radioimmunoassay, a byproduct of collagen III synthesis (PIIINP) and peptides resulting from collagen I synthesis (PINP) and degradation (ICTP) were measured. The patients with HCM were divided into two groups according to the median value of the PINP/ICTP ratio in the group. Results: At rest, the mitral annular early diastolic velocity (E’) was lower and the E/E’ was higher in the patients with HCM with high a PINP/ICTP ratio compared with patients with HCM with a low PINP/ICTP ratio and controls (p<0.001, p=0.012). With exercise, the Doppler parameters were increased in all groups, but there was no significant difference in the change in E’ and E/E’ during exercise according to collagen turnover markers. Conclusion: A higher PINP/ICTP ratio was associated with resting diastolic dysfunction in patients with HCM; however, there was no relationship with augmented diastolic dysfunction during exercise. We suggest that the type I collagen synthesis-to-degradation ratio is a useful marker of resting diastolic function in patients with HCM. Background and Objectives: Recent studies have demonstrated the usefulness of biochemical markers of collagen turnover as markers of myocardial fibrosis in various diseases. In this study, we hypothesized that increased collagen markers in patients with hypertrophic cardiomyopathy (HCM) were correlated with diastolic function at rest and diastolic functional reserve during exercise. Subjects and Methods: Thirty-six patients with HCM and 21 controls with normal left ventricular thickness were studied. Mitral septal annular velocities and mitral inflow velocities were measured at rest and during graded supine bicycle exercise (25 W, 3-minute increments) for the assessment of diastolic function at rest and during exercise. By radioimmunoassay, a byproduct of collagen III synthesis (PIIINP) and peptides resulting from collagen I synthesis (PINP) and degradation (ICTP) were measured. The patients with HCM were divided into two groups according to the median value of the PINP/ICTP ratio in the group. Results: At rest, the mitral annular early diastolic velocity (E’) was lower and the E/E’ was higher in the patients with HCM with high a PINP/ICTP ratio compared with patients with HCM with a low PINP/ICTP ratio and controls (p<0.001, p=0.012). With exercise, the Doppler parameters were increased in all groups, but there was no significant difference in the change in E’ and E/E’ during exercise according to collagen turnover markers. Conclusion: A higher PINP/ICTP ratio was associated with resting diastolic dysfunction in patients with HCM; however, there was no relationship with augmented diastolic dysfunction during exercise. We suggest that the type I collagen synthesis-to-degradation ratio is a useful marker of resting diastolic function in patients with HCM.

사람의 피부 섬유아세포 및 중간엽 줄기세포에서 항산화 활성의 반응에 대한 비교 분석

공지원,박력,박준우,이주영,최연주,문선하,김현지,전병균 한국응용과학기술학회 2019 한국응용과학기술학회지 Vol.36 No.2

The cellular senescence may be due to damage by the reactive oxygen species (ROS). This study has compared the antioxidant activity in the human cell lines of various origins, including 10S and 50S-derived normal skin fibroblasts, and 10S bone marrow, dental tissue and adipose-derived adult stem cells. After being exposed to H 2 O 2 , half inhibitory concentration (IC 50 ) values by cytotoxicity assay was significantly (P<0.05) lower in 50S-derived skin fibroblasts, than in 10S-derived skin fibroblasts and various adult stem cell lines. The cell population doubling time (PDT) and the cell frequency with high senescence associated-β-galactose activity were remarkably increased in 50S-derived fibroblasts exposed to 50 ppm H 2 O 2 for 7 days, than those of 10S-derived fibroblasts and various adult stem cell lines. Further, the expression level of antioxidant-related genes, glutathione peroxidase (GPX) and catalase (CAT), was investigated in 10S and 50S-derived skin fibroblasts, and 10S-derived various adult stem cells by reverse transcription polymerase chain reaction (RT-PCR). The expression level of GPX was higher in most of cell lines, compared to CAT, and a significantly (P<0.05) higher expression level of GPX was observed in 10S-derived skin fibroblasts and adult stem cell lines, compared to 50S-derived skin fibroblasts. We concluded that old-aged skin fibroblasts seemed to be less resistant against ROS than young-aged skin fibroblasts and adult stem cells. 본 연구는 사람의 다양한 세포주를 이용하여 활성산소종(과산화수소수)이 세포의 노화에 미치는 영향을 비교 조사하였다. 여러 농도의 과산화수소수에 세포주를 일주일 동안 배양하여 MTT 방법 으로 과산화수소수에 대한 세포 성장의 반억제농도를 구하였다. 그 결과, 50대에서 유래하는 피부 섬유 아세포와 10대의 노화 유도 피부 섬유아세포와 비교하여 10대에서 유래하는 피부 섬유아세포에서 과산 화수소수에 대한 반억제농도의 값이 유의적으로 더 높았고, 10대의 피부 섬유아세포보다는 10대의 여러 조직 기원하는 성체줄기세포에서 반억제농도의 값이 유의적으로 더 높게 관찰되었다. 또한, 50 ppm 과산화수소수를 1주일 동안 처리한 후, 50대의 피부 섬유아세포에서 다른 세포주에 비해 세포 성장이 현저히 억제되었고, 노화 관련 베타-갈락토시다아제의 활성이 증가되는 것을 관찰하였다. 또한, 활성산소의 세포 독성을 중화시키는 두 유전자, 글루타티온 과산화효소(GPX)와 카탈라아제(CAT)의 발현을 각세포주에서 조사하였을 때, CAT의 발현은 모든 세포주에서 대체로 낮았지만, GPX 유전자의 발현이 50 대의 피부 섬유아세포보다 10대의 피부 섬유아세포와 성체줄기세포에서 현저히 높게 발현되는 것을 관찰하였다. 이상의 결과에서 활성산소는 세포 노화를 유도하고, GPX의 발현이 높은 10대의 피부 섬유아 세포와 줄기세포보다는 50대의 피부 섬유아세포와 노화된 피부 섬유아세포에서 활성산소종에 대해 더큰 민감성을 가지고 있는 것을 알 수 있었다.