Various insight on biological research: analysis through bio-data of cells treated with 2′-fucosyllactose, exogenous nitric oxide on the possibility of wound repair, and ovarian protection on chemotherapy
- 주제(키워드) Bio data , Nitric oxide , GV1001
- 주제(DDC) 570
- 발행기관 아주대학교
- 지도교수 김미란
- 발행년도 2023
- 학위수여년월 2023. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000032978
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Among the human milk oligosaccharides (HMOs), many studies have been conducted on 2-fucosyllactose (2-FL), the most dominant component of HMO, due to its active and non-toxic characteristics. Despite a spotlight on the regulatory effect of 2-FL in immune response, the role of 2-FL in physiological and cellular functions associated with the human skin trouble such as hyperpigmentation has not been elucidated yet. Here, we identified a total of 1,151 DEGs changed by 2-FL in human melanoma cell line (MNT-1) through workflow of our RNA sequencing analysis. Furthermore, comparative transciptome analysis showed that up- and down-stream cellular processes and pathways related to AMPK signaling were also regulated by 2-FL treatment. Thus, we suggest the direction of future research on possibility of 2-FL as a new anti-melanogenic agent for hyperpigmentation through our network model with AMPK signaling. Nitric oxide (NO) is a free radical having an unpaired electron and is a very important molecule in many biological processes including cell proliferation and immune system. However, exogenous nitric oxide (NO) generated through plasma system is not intensively explored on various biological fields such as wound healing. Wound healing is a series of process in which the skin or tissues, repair themselves after injury. Especially, collagen and TGF-beta1 play a major role in tissue remodeling during wound healing. Here, we have manufactured a plasma generating device that can selectively produce nitric oxide (NO), exposed human fibroblast cells (adult and neonatal) to nitric oxide (NO), and showed that the expression of pErk, pSmad3, collagen, and TGF-beta1 were increased after nitric oxide (NO) treatment. However, PD98059, a specific inhibitor of mitogen-activated protein kinase (MEK1), inhibited ERK activated by nitric oxide (NO), and consequently attenuated pSmad3 and collagen. Therefore, we suggest that exogenous nitric oxide (NO) regulates collagen and TGF-beta1 through ERK-SMAD axis. Premature ovarian failure (POF) that could result from chemotherapy applied to young female cancer patients is a significant challenge in reproductive biology. A better understanding on hyperactivation of folliculogenesis or dormant primordial follicles following chemotherapy leading to problem like POF is important to adopt new strategy to maintain fertility in these patient. We provide evidence that supports that GV1001, an immunotherapeutic peptide targeting telomerase, plays a role in the protection of ovarian function through regulation of location of Foxo3a. Here, we showed that anti-VEGFa agent, bevacizumab, induced follicular loss by accelerating primordial follicle growth into primary or secondary follicles concomitant with a decline of serum anti-mullerian hormone (AMH) level and disappearance of Foxo3a signaling in the nucleus as evidenced by immunohistochemical and immunofluorescent assessment. However, GV1001 as a combinatorial administration drug reduced overactivation of folliculogenesis, thereby maintaining ovarian reserve. From this study, we suggest that GV1001 shows potential as a drug protecting from primordial follicle depletion, which may be able to maintain fertility in young cancer patients without hampering the chemical agent. Recently, biological research is changed a method centered on cell or animal experiments into a comprehensive and convergent way of thinking including bioinformatics. Also, it is a trend to obtain more efficient and progressive research results by combining genome analysis and experiments on a wide range of biological or medical problems that are difficult to see or understand experimentally in a large amount of information. In conclusion, we improved research efficiency by using a complex approach such as cell or animal experiment, and bioinformatics. Moreover, based on this approach, it is expected to be used in various fields of biology.
more목차
CHAPTER 1. Network analysis of RNA sequencing in human melanoma cells (MNT-1) through treating with 2'-fucosyllactose 1
1.1. INTRODUCTION 2
1.2. MATERIALS AND METHODS 3
1.2.1 Cell culture and materials 3
1.2.2 RNA sequencing data 3
1.2.3 Identification of differentially expressed genes (DEGs) 4
1.2.4 Functional enrichment analysis and molecular network model 4
1.3. RESULTS 6
1.3.1 Data for analysis is produced from workflow of RNA sequencing data on human melanoma cells (MNT-1) 6
1.3.2 2'-FL occurs change of cellular genes at a systematic analysis 6
1.3.3 Transcriptome analysis proposes 2'-FL-related signaling model 7
1.4. DISCUSSION 8
1.5. FIGURES LEGENDS 10
1.6. REFERENCES 16
CHAPTER 2. Exogenous nitric oxide generated through plasma system promotes collagen expression in human skin fibroblasts by increasing TGF-beta1 via ERK/SMAD pathway 25
2.1. INTRODUCTION 26
2.2. MATERIALS AND METHODS 28
2.2.1 Cell culture and cell viability assay 28
2.2.2 Nitric oxide (NO) generation and quantification 28
2.2.3 Reverse transcription polymerase chain reaction (RT-PCR) 29
2.2.4 Immunoblot assay 29
2.2.5 Immunofluorescence 30
2.2.6 ELISA assay 31
2.2.7 Collagen assay 32
2.2.8 Statistical analysis 32
2.3. RESULTS 33
2.3.1 Microwave plasma system and concentration of nitric oxide (NO) for experiment 33
2.3.2 Viability of cell on nitric oxide (NO) generated various O2 flow rates 33
2.3.3 Levels of collagen 1 type and TGF-beta1 gene increase in human fibroblast cells (adult and neonatal) treated with nitric oxide (NO) 34
2.3.4 Nitric oxide (NO) regulates phosphorylation and translocation of SMAD3 through ERK signaling 35
2.3.5 Nitric oxide (NO) leads to the promotion of collagen by induction of TGF-beta1 through ERK-SMAD3 axis 36
2.4. DISCUSSION 37
2.5. FIGURES LEGENDS 39
2.6. REFERENCES 54
CHAPTER 3. GV1001 protects ovarian function from over-activation of folliculogenesis triggered by an anti-VEGFa agent through VEGFa/AMPK/Foxo3a pathway 59
3.1. INTRODUCTION 60
3.2. MATERIALS AND METHODS 63
3.2.1 Animal 63
3.2.2 Ovarian histology and follicle counting 64
3.2.3 Immunoblot assay 65
3.2.4 Immunohistochemistry assay 66
3.2.5 Immunofluorescence 67
3.2.6 ELISA assay 67
3.2.7 Statistical analysis 68
3.3. RESULTS 69
3.3.1 GV1001 reduces loss of follicle by accelerating follicles activation and growth through anti-VEGFa agent (Bevacizumab) 69
3.3.2 GV1001 deters follicle loss induced by anti-VEGFa agent (Bevacizumab) 71
3.3.3 Co-administration of GV1001 and anti-VEGFa agent (Bevacizumab) maintain the level of AMH to be important as indicator of follicle reserve 71
3.3.4 GV1001 leads to translocation of Foxo3a (forkhead transcription factor) limited by anti-VEGFa agent (Bevacizumab) 72
3.4. DISCUSSION 74
3.5. FIGURES LEGENDS 77
3.6. REFERENCES 91
ABSTRACT (Korean) 95
