성체줄기세포 치료 후 뇌 경색 쥐 모델 뇌 조직에서 기체크로마토그래피-질량분석기를 이용한 유기산 프로파일 분석
Organic acid profiling analysis by gas chromatography – mass spectrometry in ischemic brain of rat model after mesenchymal stem cells therapy
- 주제(키워드) Organic acids , Methoximation/tert-butyldimethylsilylation , Gas chromatography-mass spectrometry , Stroke , Human mesenchymal stem cell , Star pattern
- 발행기관 아주대학교
- 지도교수 이광
- 발행년도 2009
- 학위수여년월 2009. 8
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000010121
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Mesenchymal stem cell (MSC) may have the potential to improve recovery and promote brain repair after stroke. However, therapeutic effect monitoring of biochemical compounds following transplantation of human MSC (hMSC) into stroke animal model have not been reported. To examine how organic compounds change in brain after transplantation of hMSC in rat that had undergone transient middle cerebral artery occlusion (MCAo), we investigated the content of organic acids (OAs), which are important biochemical indicators in pathological conditions. An efficient method for the simultaneous profiling analysis of organic acids (OAs) including keto acids (KAs) as their methoximation/tert.-butyldimethylsilylation (MO/TBDMS) derivatives was developed by gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode. In this study, the keto groups of KAs were reacted with methoxyamine hydrochloride for the simultaneous analysis of 21 OAs including 3-hydroxybutyric acid. Then the aqueous solution was acidified with concentrated sulfuric acid (pH ≤ 2), followed by solvent extraction with diethyl ether and ethyl acetate in sequence. The combined extracts were evaporated to dryness under a gentle stream of nitrogen. The remaining active hydrogen atoms were converted to TBDMS derivatives for the direct analysis by GC-SIM-MS. Under the optimal conditions, linear response in the range of 0.01-5.0 μg/mL was achieved with good correlation coefficient (r ≥ 0.995). The precision showed low relative standard deviation less than 10 %, and the accuracy (% RE) varied from -8.0 to 6.7 for 21 OAs studied. When applied to rat brain tissues from normal control group, MCAo group and MCAo group following intravenous hMSC transplantation, the levels of some OAs of the MCAo and MCAo + hMSC groups were significantly different from those of the control group. In addition, normalized OA levels of MCAo and MCAo + hMSC groups were transformed into distorted star patterns which were different from control mean. The present method has provided insights into understanding the complexity of biochemical and physiological events that occur in ischemic brain injury and the transplantation effects of MSCs in stroke.
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ABSTRACT ⅰ
CONTENTS ⅲ
LISTS OF TABLE ⅴ
LISTS OF FIGURE ⅵ
Ⅰ. INTRODUCTION 1
Ⅱ. MATERIALS AND METHODS 5
1. Chemicals and reagents 5
2. Preparation of standards solutions 5
3. Gas chromatography–mass spectrometry 8
4. Derivatization and solvent extraction 10
5. Method validation for assay of organic acids 13
6. Sample preparation for assay of organic acids in rat brain tissues 13
7. Star symbol plotting 14
Ⅲ. RESULTS 16
1. Simultaneous profiling analysis of 21 organic acids 16
2. Mass spectral properties of organic acids as MO/TBDMS derivatives 16
3. Method validation for simultaneous assay of 21 organic acids 19
4. Levels of organic acids in rat brain tissue 23
5. Star pattern recognition analysis 28
Ⅳ. DISCUSSION 33
Ⅴ. CONCLUSION 35
REFERENCES 36
국문요약 41
