임신초기 돼지 영양외배엽 세포의 발달에 미치는 EGF, IGF-I, VEGF 및 CSF2의 기능분석 연구

학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부(바이오모듈레이션전공), 2014. 2. 안용준.The majority of early conceptus mortality in pregnancy occurs during the peri-implantation stage, suggesting that this period is important for conceptus viability and the establishment of pregnancy. Successful establishment of pregnancy in all mammalian species depends on the orchestrated molecular events that transpire at the conceptus-uterine interface during the peri-implantation phase. This maternal-conceptus interaction is especially crucial in pigs because in them non-invasive epitheliochorial placentation occurs, in which the pre-implantation phase is prolonged. During the pre-implantation period, conceptus survival and the establishment of pregnancy are known to depend on the developing conceptus receiving an adequate supply of histotroph, which contains a wide range of nutrients and growth factors. Evidence links epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), vascular endothelial growth factor (VEGF), and colony-stimulating factor 2 (CSF2) to embryogenesis or implantation in various mammalian specieshowever, in the case of pig, little is known about such functions of these growth factors, especially their regulatory mechanisms at the maternal-conceptus interface. Therefore, the objectives of this study were to determine: 1) the temporal and cell-specific expression of EGF, IGF-I, VEGF, and CSF2 signaling systems in the porcine endometrium during the estrous cycle and early pregnancy2) the potential intracellular signaling pathways responsible for the activities of these four factors in primary porcine trophectoderm (pTr) cellsand 3) the changes in cellular activities induced by these promising factors. First, the functional effect and cellular signaling cascades in pTr cells induced by EGF, which exhibits potential growth-promoting activities on the conceptus and endometrium, were investigated. EGFR mRNA and protein were abundant in endometrial luminal epithelia (LE) and glandular epithelia (GE), stratum compactum stroma, and conceptus trophectoderm on Days 13-14 of pregnancy, but not in any other cells of the uterus. EGF treatment of pTr cells increased the abundance of phosphorylated (p)-AKT1, p-ERK1/2 MAPK and p-P90RSK in the nucleus and/or cytoplasm when compared with the levels in control cells. Furthermore, EGF-stimulated phosphorylation of AKT1 and ERK1/2 MAPK were inhibited in pTr cells transfected with an EGFR siRNA, and compared with control siRNA-transfected pTr cells, the EGFR siRNA-transfected pTr cells exhibited an increase in the expression of gene encoding interferon (IFN)-δ and transforming growth factor (TGF) β-1by contrast, no effect was detected on the expression of the gene encoding IFN-γ. Moreover, EGF stimulated the proliferation and migration of pTr cells, but these stimulatory effects were blocked by pharmacological inhibitors such as SB203580 (a p38 inhibitor), U0126 (a MAPK inhibitor), rapamycin (an MTOR inhibitor), and LY294002 (a PI3K inhibitor). Second, IGF-I was examined. IGF-1 is another promising growth factor that is known to play key roles in reproductive processeshowever, little is known about IGF-I-induced functional effects and regulatory mechanisms during peri-implantation in pigs. In this study, endometrial type I IGF receptor (IGF-IR) mRNA was determined to increase substantially during early pregnancy relative to the level during the estrous cycle, and the mRNAs of both IGF-I and IGF-IR were abundant in endometrial LE and GE, stroma and conceptus trophectoderm on Day 12 of pregnancy. Moreover, IGF-I treatment potently increased the amounts of p-AKT1 and, ERK1/2 MAPK in the nucleus and cytoplasm and of RPS6 in the cytosol when compared with the amounts in untreated pTr cells, and IGF-I-induced activation of AKT1 and ERK1/2 was blocked by LY294002. Furthermore, IGF-I strongly stimulated both the proliferation and the migration of pTr cells, but these effects were inhibited by SB203580, U0126, rapamycin and LY294002. Third, this study focused on VEGF, which was identified as a potential mediator of the fetal-maternal dialog that regulates the development of the peri-implantation porcine conceptus. In addition to its known angiogenic effects, VEGF has been suggested to play roles in the development of the early embryo, but VEGF-induced effects on the peri-implantation conceptus remain unknown. Results of this study revealed that endometrial VEGF, VEGF receptor (VEGFR)-1, and VEGFR-2 mRNA levels in endometrial LE and GE, endothelial blood vessels, and scattered cells in the stroma were more abundant during the peri-implantation period of pregnancy than during the estrous cycle. Moreover, VEGF treatment of pTr cells increased the abundance of p-AKT1, p-ERK1/2, p-p70RSK, p-RPS6 and p-4EBP1, and the addition of LY294002 suppressed VEGF-induced phosphorylation of ERK1/2 and AKT1. Furthermore, VEGF potently stimulated both the proliferation and the migration of pTr cells, but these effects were inhibited in the presence of SB203580, U0126, rapamycin and LY294002. The fourth promising cytokine studied was CSF2, which is also known as granulocyte-macrophage colony-stimulating factor (GM-CSF). CSF2 plays a role in facilitating mammalian early embryonic development. In this study, endometrial CSF2 mRNA expression was determined to be increased during the peri-implantation period relative to the mRNA level during the estrous cycle. In pTr cells, CSF2 significantly induced the activation of AKT1, ERK1/2, MTOR, p70RSK, and RPS6, but not of STAT3, and the addition of LY294002 abolished CSF2-induced increases in p-ERK1/2, p-MTOR, and p-AKT1 levels. Furthermore, CSF2 strongly stimulated pTr cell proliferation, an effect that was blocked by U0126, rapamycin and LY294002. Collectively, these results provide new insights into the potential mediators that regulate the development of the peri-implantation conceptus at the fetal-maternal interface. These results indicate that endometrial- and/or conceptus derived EGF, IGF-I, VEGF, and CSF2 critically affect the growth and development of porcine trophectoderm cells, and that these stimulatory effects are coordinately regulated by multiple cellular signaling cascades including the PI3K-AKT and ERK1/2 MAPK pathways during early pregnancy in pigs.CHAPTER 1. General Introduction 1 CHAPTER 2. Literature Review 6 1. Estrous Cycle and Early Pregnancy 7 1.1. Estrous Cycle 7 1.2. Pre-Implantation Period 9 1.3. Opening of the Implantation Window and Implantation 10 2. Physiology of the Porcine Conceptus during Peri-Implantation 12 2.1. Early Conceptus Development 12 2.2. Maternal Recognition of Pregnancy 12 2.3. Conceptus Trophoblastic Elongation 13 2.4. Conceptus Apposition and Attachment to the Uterine Luminal Epithelium 15 3. Uterine Microenvironment during Early Pregnancy 16 3.1. Maternal-Conceptus Interactions 16 3.2. Embryonic Factors 17 3.3. Uterine Factors 18 4. Growth Factors and Cytokines Regulating Conceptus Cellular Processes 19 5. Promising Uterine Factors Affecting Conceptus Development and Implantation 21 5.1. Epidermal Growth Factor (EGF) 21 5.2. Insulin-Like Growth Factor-I (IGF-I) 22 5.3. Vascular Endothelial Growth Factor (VEGF) 23 5.4. Colony-Stimulating Factor 2 (CSF2) 24 CHAPTER 3. Epidermal Growth Factor Stimulates Proliferation and Migration of Porcine Trophectoderm Cells Through Protooncogenic Protein Kinase 1 and Extracellular-Signal-Regulated Kinases 1/2 Mitogen- Activated Protein Kinase Signal Transduction Cascades during Early Pregnancy 27 1. Abstract 28 2. Introduction 30 3. Materials and Methods 34 4. Results 44 5. Discussion 61 CHAPTER 4. Insulin-Like Growth Factor I Induces Proliferation and Migration of Porcine Trophectoderm Cells through Multiple Cell Signaling Pathways, Including Protooncogenic Protein Kinase 1 and Mitogen- Activated Protein Kinase 67 1. Abstract 68 2. Introduction 70 3. Materials and Methods 74 4. Results 82 5. Discussion 96 CHAPTER 5. Stimulatory Effect of Vascular Endothelial Growth Factor on Proliferation and Migration of Porcine Trophectoderm Cells and Their Regulation by the Phosphatidylinositol-3-Kinase-AKT and Mitogen- Activated Protein Kinase Cell Signaling Pathways 103 1. Abstract 104 2. Introduction 106 3. Materials and Methods 110 4. Results 118 5. Discussion 133 CHAPTER 6. Proliferation-Stimulating Effect of Colony Stimulating Factor 2 on Porcine Trophectoderm Cells is Mediated by Activation of Phosphatidylinositol 3-Kinase and Extracellular Signal-Regulated Kinase 1/2 Mitogen-Activated Protein Kinase 139 1. Abstract 140 2. Introduction 142 3. Materials and Methods 146 4. Results 152 5. Discussion 162 CHAPTER 7. AHCYL1 Is Mediated by Estrogen-Induced ERK1/2 MAPK Cell Signaling and MicroRNA Regulation to Effect Functional Aspects of the Avian Oviduct 168 1. Abstract 169 2. Introduction 170 3. Materials and Methods 173 4. Results 182 5. Discussion 202 CHAPTER 8. Paradoxical Expression of AHCYL1 Affecting Ovarian Carcinogenesis between Chickens and Women 208 1. Abstract 209 2. Introduction 211 3. Materials and Methods 213 4. Results 222 5. Discussion 241 CHAPTER 9. Cell-Specific and Temporal Aspects of Gene Expression in the Chicken Oviduct at Different Stages of the Laying Cycle 245 1. Abstract 246 2. Introduction 247 3. Materials and Methods 249 4. Results 254 5. Discussion 269 CHAPTER 10. Recrudescence Mechanism and Gene Expression Profile of the Reproductive Tracts in Chicken during the Molting Period 276 1. Abstract 277 2. Introduction 279 3. Materials and Methods 283 4. Results 292 5. Discussion 314 CHAPTER 11. Conclusion 323 REFERENCES 331 초록 376Docto

Evaluation of Social Nicotine Dependence Using the Kano Test for Social Nicotine Dependence (KTSND-K) Questionnaire in Korea

BACKGROUND: Smoking is one of the most important leading causes of morbidity and mortality. Smoking habit is recognized as nicotine dependence, which consists of physical and psychosocial dependence. To evaluate social nicotine dependence, the Kano Test for Social Nicotine Dependence (KTSND) working group developed a new questionnaire, which consists of 10 questions with a total score of 30 in Japan. We examined the social nicotine dependence among healthy adults using the new KTSND questionnaire and evaluated validity of the KTSND questionnaire in Korea. METHOD: We applied Korean KTSND questionnaire version 2 to employees of hospital, university students and people for medical examination and promotion test. Complete data obtained from the 741 responders were analyzed. RESULT: The mean age of responders was 31.8 years. Among them, males were 57.8%. Current smokers, ex-smokers, and non-smokers were 13.8%, 12.8%, and 73.4% respectively. According to smoking status, the total KTSND scores of current smokers were significantly higher than those of ex-smokers, and of non-smokers (17.1+/-5.4 versus 14.3+/-5.5, and 12.3+/-5.5, p or =4), This finding suggested that the psychosocial dependence might play a different role from physical nicotine dependence in smoking. Most of the non-smokers (62.5%) had an experience of harmful passive smoking especially in public place. CONCLUSION: Our study suggested that the KTSND questionnaire could be a useful method to evaluate psychosocial aspects of smokingope

A Case of Pulmonary Metastasis of a Dermatofibrosarcoma Protuberasn

Dermatofibrosarcoma protuberans (DFPS) is a locally aggressive skin tumor with a very low incidence in the general population. This tumor has a remarkable tendency to recur, However, a metastasis is rare. We report a case of DFPS with a pulmonary metastasis in 28-year-old man. The pulmonary metastasis developed 5 years after a complete resection of the primary skin tumor. We reviewed the clinical manifestations and treatment of DFPS, and highlight the need for a long-term follow-up examination for metastases after a wide excision of these lesions.ope

Applicability of American and European Spirometry Repeatability Criteria to Korean Adults

Background: The objective of this study was to evaluate the clinical applicability of the repeatability criteria recommended by the American Thoracic Society/European Respiratory Society (ATS/ERS) spirometry guidelines and to determine which factors affect the repeatability of spirometry in Korean adults. Methods: We reviewed the spirometry data of 4,663 Korean adults from the Korean National Health and Nutritional Examination Survey (KNHANES) Chronic Obstructive Pulmonary Disease Cohort (COPD cohort) and the Community-based Cohort Study VI-Fishing village/Islands (community cohort). We measured the anthropometric factors and differences between the highest and second-highest FVC (dFVC) and FEV1 (dFEV1) from prebronchodilator spirometry. Analyses included the distribution of dFVC and dFEV1, comparison of the values meeting the 1994 ATS repeatability criteria with the values meeting the 2005 ATS/ERS repeatability criteria, and the performance of linear regression for evaluating the influence of subject characteristics and the change of criteria on the spiro-metric variability. Results: About 95% of subjects were able to reproduce FVC and FEV1 within 150 ml. The KNHANES based on the 1994 ATS guidelines showed poorer repeatability than the COPD cohort and community cohort based on the 2005 ATS/ERS guidelines. Demographic and anthropometric factors had little effect on repeatability, explaining only 0.5 to 3%. Conclusion: We conclude that the new spirometry repeatability criteria recommended by the 2005 ATS/ERS guidelines is also applicable to Korean adults. The repeatability of spirometry depends little on individual characteristics when an experienced technician performs testing. Therefore, we suggest that sustained efforts for public awareness of new repeatability criteria, quality control of spirograms, and education of personnel are needed for reliable spirometric results.ope

A Case of Successful Management of Sarcoidosis with Chylothorax Using Octreotide

Sarcoidosis is a multi-system granulomatous disorder of an unknown etiology and affects individuals worldwide. It is characterized pathologically by the presence of non-caseating granulomas in more than one involved organ. However, pleural involvement of sarcoidosis is rare and there are no reported cases in Korea. Traditionally, sarcoidosis has often been treated with systemic corticosteroids or cytotoxic agents. In particular, chylothorax with sarcoidosis is usually treated with corticosteroid for approximately 3~6 months, followed by repeated therapeutic thoracentesis, talc pleurodesis, dietary treatment, or thoracic duct ligation where needed. We encountered a 46 years old female patient presenting with cough, dyspnea and both hilar lymphadenopathy (stage I) on chest radiograph. The patient was diagnosed with a non-caseating granuloma, sarcoidosis by a mediastinoscopic biopsy. For one month, she had suffered from dyspnea due to right side pleural effusion, which was clearly identified as a chylothorax on thoracentesis. Corticosteroid therapy with dietary adjustment was ineffective. She was treated successfully with a subcutaneous injection of octreotide for 3 weeks and oral corticosteroid. We report a case of successful and rapid treatment of chylothorax associated with sarcoidosis using octreotide and oral corticosteroid.ope

A Case of Pleural Effusion due to Hypothyroidism

Serous effusions have been most commonly associated with ascites, pericardial effusion, and heart failure. But, they have been considered to be an unusual form of complication in hypothyroidism and pleural effusion, which has been observed as an isolated finding in hypothyroidism is apparently rare and complete analysis of these types of hypothyroid-associated pleural effusions has yet not been described. We report a case of hypothyroidism associated with unilateral pleural effusion in a 77 year-old male patient who was improved through levothyroxine sodium with brief review of the literature.ope

The Relationship between Age and Pleural Fluid Adenosine Deaminase Activity in Pleural Tuberculosis

연구 배경 : Adenosine deaminase (ADA)는 퓨린 (purine) 대사에 작용하는 효소로서 림프구, 특히 T-림프구의 증식과 분화에 관여하며,결핵성 흉수의 진단에 있어서 중요한 생화학적 표지자 중의 하나이다.한편, 노인의 경우, T-림프구의 수와 기능의 감소에 의하여 면역 기능이 감소하는 것으로 알려져 있다. 이에 저자 등은 노인 결핵성 흉수 환자에서 흉수내의 ADA 수치가 젊은 환자에서보다 감소하는지를 조사하였다. 방 법 : 4년 동안 세브란스 병원에서 1) 흉수 결핵균 배양 양성 또는 2) 흉막 조직 검사상 결핵에 합당한 소견을 보여 결핵성 흉수로 진단받은 환자 80명을 대상으로 후향적으로 조사하였다. 65세를 기준으로 두 군으로 분류하였으며, 연령과 흉수 ADA 수치의 연관 관계를 독립 표본 t-검정 및 선형 회귀 분석을 이용하여 연구하였다. 결 과 : 80명의 환자 중 65세 이상은 21명 (26.3%)이었다. 흉수 내의 ADA 수치는 65세 이상 및 이하 군에서 각각 71.2± 27.6 IU/L, 68.5 ± 25.8 IU/L 이었다 (p=0.69). 선형 회귀 분석에서도 연령과 흉수 내의 ADA 수치는 상관 관계를 보이지 않았다 (r²=0.05, p=0.59). 결 론 : 본 연구의 결과에 의하면,결핵성 흉수의 진단에서 흉수 ADA 수치를 보조 지표로 사용하는 데 있어서, 노인환자에서도 젊은 환자와 동일한 임상적 유의성을 가지고 동일한 결정 수치 (cut-off value)를 적용할 수 있을 것으로 판단된다.ope

A Case of Pneumothorax Caused by Cystic Lung Metastasis of Angiosarcoma

Angiosarcoma is a rare but highly malignant tumorthat usually arises in the scalp or face of elderly males. Distant metastases favor the lung, liver, lymph nodes and skin. Metastatic pulmonary angiosarcoma commonly takes the form of a nodule but can sometimes appear as a thin-walled cyst. We report a case of 65 years-old male with a spontaneous pneumothorax, who underwent excision and radiotherapy for an angiosarcoma of the scalp 2 years ago. A chest CT scan revealed multiple cysts in the lung. The video-assisted thoracoscopic lung biopsy demonstrated subpleural cysts without tumor cells. A skin biopsy of the scalp showed an angiosarcoma. This case was diagnosed as a recurrence of an angiosarcoma with a supposed lung metastasis. This case suggests that a spontaneous pneumothorax in elderly people may be secondary to a pulmonary metastasis from an angiosarcoma of the scalp.ope

A Case of Pulmonary Sarcoidosis Combined with Neurosarcoidosis

Sarcoidosis is a multi-systemic syndrome of an unknown etiology, and it is characterized by the formation of multiple noncaseating granulomas that disrupt the architecture and function of the tissues in which they reside. The most commonly affected organs are lung, skin and lymph nodes. Overt clinical involvement of the nervous system is uncommon and this occurs in about 5% of all patients during the course of their disease. The most common manifestations are granulomatous leptomeningitis, cranial nerve palsy, electrolyte or other endocrinologic abnormalities, but isolated memory impairment is a rare manifestation. This is a case of 59 years-old male with recent memory impairment, and he was previously diagnosed with pulmonary sarcoidosis by transbronchial lung biopsy. The brain MRI imaging revealed the leptomeningeal and parenchymal involvement of sarcoidosis. He was treated with high dose corticosteroid and his memory function was improved to nearly a normal level. We report here on a case of successful treatment of pulmonary sarcoidosis combined with neurosarcoidosis with using high dose corticosteroid, and the patient presented with recent memory impairment.ope

A Case of Pulmonary Choriocarcinoma

Pulmonary choriocarcinoma is a very rare tumor in men. Herrein, the case of a pulmonary choriocarcinoma in 39-year-old man, and whether it had a primary nature, is reported. He denied any prior medical illness, but was admitted to our hospital with a history of a cough, and progressive dyspnea and hemoptysis 2 and 1 week duration, respectively. Chest radiographs on admission revealed a huge lung mass, 10 cm in diameter, in the left upper lung field, with left pleural effusion. Although biopsies using several diagnostic methods for the pathological confirmation were attempted, the pathology was not confirmed. Finally, the patient died after 2 months of regression. An autopsy of the lung was then performed.ope