돼지 난자의 체외성숙과 단위발생시 7,8-Dihydroxyflavone의 효과

학위논문 (석사)-- 서울대학교 대학원 : 수의학과(수의산과학전공), 2013. 2. 이병천.One of the factors that impair the in vitro produced porcine embryos is the oxidative stress that mainly caused by the imbalance between reactive oxygen species (ROS) generation and antioxidants activity, especially glutathione (GSH). Here, the effect of 7,8-dihydroxyflavone (7,8-DHF), a flavonoid antioxidant, on porcine oocyte maturation and its developmental competence was examined. Porcine oocytes were cultured in media supplemented with 0, 1, 5, and 10 μM 7,8-DHF during both in vitro maturation (IVM) and in vitro culture (IVC) after parthenogenetic activation. Maturation of oocytes was evaluated based on the 1st polar body (PB) extrusion and intracellular GSH level and developmental competence was assessed through observing cleavage and blastocyst formation. In each step, the levels of intracellular GSH and ROS were assessed by fluorescence intensity and the apoptosis-related gene expression was examined using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Parthenogenetic mbryos treatment with 1 μM 7,8-DHF during IVM and IVC showed increased cytoplasmic maturation and reached blastocysts stage (36.1 ± 3.5%) at a higher rate than the other groups (24.7 ± 3.1, 16.0 ± 2.6, and 10.3 ± 2.2%, P < 0.05). In that group, intracellular GHS level was significantly increased after IVM and IVC and ROS was decreased after IVC (P < 0.05). Moreover, it showed high expression of anti-apoptotic gene (BCL2L1) and low expression of pro-apoptotic gene (BAK1) (P < 0.05). In conclusion, 1 μM 7,8-DHF treatment during IVM and IVC showed an anti-apoptotic effect by increasing intracellular GSH synthesis and scavenging ROS, therefore, it improves the developmental competence of porcine parthenogenetic embryos.ABSTRACT i TABLE OF CONTENTS iii LIST OF TABLES iv LIST OF FIGURES v PUBLICATION LISTS vi LITERATURE REVIEW 1 1. Redox system in oocytes and embryos 2 2. Antioxidants for oocytes and embryos 5 3. Parthenogenesis 8 INTRODUCTION 10 MATERIALS AND METHODS 13 RESULTS 22 DISCUSSION 34 REFERENCES 39 국문초록 50Maste

사람 Telomerase 역전사효소 서열 유래 펩타이드 GV1001의 항염 효능에 대한 연구

학위논문 (석사)-- 서울대학교 대학원 : 의학과(해부학 전공), 2015. 8. 강재승.GV1001 is a peptide derived from human telomerase reverse transcriptase (hTERT) sequence, and has anti-cancer and anti-inflammatory effect. Enolase1 (ENO1) is a glycolytic enzyme and its stimulation induces to produce a large amount of pro-inflammatory cytokines from concanavalin (Con) A -activated peripheral blood mononuclear cells (PBMCs) and from ENO1 expressing monocytes and macrophages from rheumatoid arthritis (RA) patients. However, it is still unknown whether GV1001 could regulate the ENO1-mediated pro-inflammatory cytokines production. Therefore, I investigated whether GV1001 regulates ENO1-mediated pro-inflammatory cytokines production as an anti-inflammatory peptide. First, I found that GV1001 does not affect the expression of ENO1 on Con A-activated PBMCs and RA PBMCs. However, ENO1 stimulation increased the production of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) from Con A-activated PBMCs. And it is down-regulated by the pre-treatment of GV1001. GV1001 also decreases the production of pro-inflammatory cytokines from ENO1 stimulated RA PBMCs. And then I examined what kinds of signaling molecules are involved in the down-regulation of ENO1-mediated pro-inflammatory cytokine production by GV1001. When ENO1 on the surface of Con A-activated PBMCs and RA PBMCs is stimulated, I found that p38 MAPK and NF-κB are activated. However, these are successfully suppressed by the pre-treatment of GV1001. Taken together, GV1001 might be a useful anti-inflammatory peptide via the down-regulation of pro-inflammatory cytokine production and the suppression of the p38 MAPK and NF-κB activation by ENO1 stimulation.CONTENT Abstract ............................................................... ⅰ Contents .............................................................. ⅳ List of Figures ................................................... ⅶ List of Abbreviations ........................................ ⅹ Introduction ......................................................... 1 Materials and Methods 1. Isolation of PBMCs .................................................................... 5 2. Stimulation of PBMCs with Con A .......................................... 5 3. Preparation of Anti-ENO1 monoclonal antibody.................... 6 4. ENO1 stimulation ....................................................................... 7 5. GV1001 treatment ..................................................................... 7 6. Flow cytometry analysis .......................................................... 7 7. Enzyme-Linked Immunosorbent Assay (ELISA) ................. 8 8. Inhibitor study for signal pathway ........................................ 9 9. Immunoblotting ........................................................................... 9 10. Statistical analysis ................................................................. 10 Results 1. Con A stimulation increases ENO1 expression on the surface of PBMCs ................................................................................................... 12 2. GV1001 doesn't affect ENO1 expression on PBMCs by Con A treatment ................................................................................................... 16 3. GV1001 decreases the production of TNF-α, IL-1β and IL-6 by ENO1 stimulation ................................................................................................... 18 4. GV1001 suppresses the activation of p38 MAPK and NF-κB in Con-A activated PBMCs: ELISA ................................................................................................... 22 5. GV1001 suppresses the activation of p38 MAPK and NF-κB in Con-A activated PBMCs: Immunoblotting ................................................................................................... 25 6. GV1001 doesn't change the expression level of ENO1 on PBMCs from RA patients ................................................................................................... 31 7. GV1001 decreases the production of TNF-α, IL-1β and IL-6 from RA PBMCs by ENO1 stimulation ................................................................................................... 34 8. GV1001 suppresses the activation of p38 MAPK and NF-κB in RA PBMCs. ................................................................................................... 37 Discussion .......................................................... 39 References ......................................................... 44 Abstracts in Korean ......................................... 52Maste