SV40 -T- antigen 으로 불사화된 중간엽 줄기세포에서의 대사체학적 분석연구
Studies on metabolomic analysis with immortalized mesenchymal stem cells by SV40 T-antigen
- 주제(키워드) amino acid , gas chromatography-mass spectrometry , hMSCs , immortalized cell line , SV40 T antigen
- 발행기관 아주대학교
- 지도교수 이광
- 발행년도 2017
- 학위수여년월 2017. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000025485
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
This study was carried out by transfection of SV40 T antigen into mesenchymal stem cell hMSCs collected from human bone marrow. Immortalized hMSCs-T cell is a mesenchymal stem cell capable of multiple cell division. The cell cycle of hMSCs was relatively short, but hMSCs-T cell was able to divide more than 80 passages. The amount of amino acids (AAs) in the cell was measured by gas chromatography in order to examine the metabolism of the cell according to the elongated cell cycle. Amino acids are the key component of protein synthesis and intracellular nitrogen. In addition, we aimed to study the amino acid alteration and find a biomarker that can support the continuous growth of cells that are capable of continuously production for the industrial use of immortalized stem cells. The alterations of amino acids in mesenchymal stem cells and immortalized stem cells are written in chapter 1. Beside hMSCs-T cell, HEK293 transfected with synphilin-1 also altered amino acid composition. Synphilin-1 is an α-synuclein interacting protein in Lewy body, which is a characteristic of Parkinson's disease. When HEK293 and S293, HEK293 stably overexpressed synphilin-1, were starved with KRBB buffer, the morphological changes and cell aggregations were different between cell types. The difference might be from intracellular metabolites and protein compositions. Amino acid sensing pathway is controlled by intracellular and extracellular amount of amino acid and changes of amino acids are important factors in controlling protein synthesis and degradation. The mammalian target of rapamycin (mTOR) is important in amino acid sensing that regulates cell growth and cell survival. This study is to estimate the amount of amino acid and detect the phosphorylation of S6K1 and Akt influenced by mTORC1 and mTORC2. HEK293 and S293 were starved and challenged with glutamine, glutamic acid, and proline to confirm the amino acid alteration induced from synphilin-1. The results of amino acid and biological data were recorded in chapter 2.
more목차
Chapter1. Studies on metabolomics analysis with immortalized mesenchymal stem cells by SV40 T-antigen 1
1.1. Abstract 2
1.2 Introduction 3
1.3. Materials and Methods 5
1.3.1. Chemicals and reagents 5
1.3.2. Cell culture and transfection 5
1.3.3. Cell preparation and cell lysis 5
1.3.4. Preparation of standard solutions 6
1.3.5. Sample preparation for amino acid assays 6
1.3.6. Gas chromatography-Mass spectrometry 7
1.3.7. Data analysis 7
1.3.8. Statistical analysis 7
1.4. Results 10
1.4.1. SIM Chromatogram of amino acid as EOC/TBDMS derivatives 10
1.4.2. Composition changes of amino acid with hydrophobic side chain in hMSCs and hMSCs-T cells 13
1.4.3. Composition changes of amino acid with negatively charged side chain in hMSCs and hMSCs-T cells 14
1.4.4. Changes of amino acids with electrically positive charged side chains and special cases in hMSCs and hMSCs-T cell. 15
Discussions 16
Chapter 2. Studies on metabolomic analysis in synphilin-1 over-expressing HEK293 cells 18
2.1. Abstract 19
2.2. Introduction 20
2.3. Materials and methods 24
2.3.1. Cell culture and transfection 24
2.3.2. Preparation of cell lysate for metabolic analysis 24
2.3. 3. Preparation of standard solutions for GC-MS. 25
2.3.4. Sample preparation for amino acid assays 25
2.3.5. Gas chromatography–mass spectrometry 25
2.3.6. Cell starvation and restimulation 26
2.3.7. Western blot analysis 26
2.3.8. Total RNA isolation and semi-quantitative RT-PCR assays 27
2.3.9. Statistical analysis 27
2.4. Results 28
2.4.1. Morphological observations for HEK293 and S293 28
2.4.2. Phosphorylation of S6K1 and Akt in HEK293 and S293 28
2.4.3. Quantification of amino acids as EOC/TBDMS derivatives 31
2.4.3.1. Changes of amino acid with hydrophobic side chain 32
2.4.3.2. Changes of amino acid with polar uncharged side chains 36
2.4.3.3. Changes of amino acid with electrically negative charged side chains 36
2.4.3.4. Changes of amino acid with electrically positive charged side chains and special cases 39
2.4.4. Time-dependent expression of phosphorylated S6K1 42
2.4.5. Dose-dependent expression of S6K1-pT389 by amino acids. 43
2.4.6. Expression of S6K1-pT389 and Akt-pS473 in HEK293 and S293 44
2.5. Discussions 47
2.6. References 50
ABSTRACT IN KOREAN 55
