뇌종양에서 중간엽줄기세포를 이용한 자살유전자치료의 개발
Development of suicide gene therapy using mesenchymal stem cells for the treatment of brain tumors
- 주제(키워드) Mesenchymal stem cells (MSCs) , Cytosine deaminase (CD) , spleen focus forming virus (FIP) , 5-Fluorocytosine (5-FC) , 5-Fluorouracil (5-FU) , Glioblasoma multiforme (GBM) , 5-fluorouracil resistance mechanism
- 발행기관 아주대학교
- 지도교수 서해영
- 발행년도 2010
- 학위수여년월 2010. 2
- 학위명 박사
- 학과 및 전공 일반대학원 신경과학기술과정
- 실제URI http://www.dcollection.net/handler/ajou/000000010527
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Gliomas are the most common primary tumors of the central nervous system. Over the past several years, despite of advanced technique in surgery, radiotherapy and chemotherapy but gliomas still remains dismal. The median survival rate after diagnosis of glioblastoma multiforme (GBM), the most common and aggressive subtype of malignant glioma, is under 1 year. (Surawicz et al. 1998; Legler et al. 1999) Although suicide genes such as cytosine deaminase (CD) have recently emerged as an attractive alternative therapy for the treatment of various types of cancers, their effectiveness of suppressing the recurrent glioblastoma has not been clearly demonstrated. In this study, I investigated critical parameters that are likely to affect the clinical efficacy of the CD gene therapy and pre-diagnostic marker in the treatment of malignant glioblastoma. Mesenchymal stem cells (MSCs) can be used as a cellular vehicle to deliver suicide gene to brain tumors. MSCs derived from human bone marrow were transduced with a replication incompetent retroviral vector encoding a bacterial cytosine deaminase (CD) that can convert non-toxic 5-fluorocytosine (5-FC) into cytotoxic 5-fluorouracil (5-FU). CD-expressing MSCs exerted strong dose-dependent cytotoxic effects on co-cultured C6 glioma cells in the presence of 5-FC. The half maximal concentration of 5-FC was 49.4 ？？M for CD-expressing MSCs (suicide effects) and 56.3 ？？M for co-cultured C6 cells (bystander effects). Intracranial transplantation of CD-expressing MSCs reduced the tumor mass derived from co-transplanted C6 cells by 95% in the rat brain tumor model after daily administration of 500 mg/kg 5-FC for two weeks. In the case of a pre-existing tumor, a single transplantation of MSC-FIP/CD and daily administration of 5-FC for three weeks, however, was not sufficient to reduce tumor mass. In contrast, multiple transplantations of CD-expressing MSCs every 7 days for 3 weeks could suppress tumor recurrence. Parameters related to 5-FU resistance were investigated for pre-diagnostic markers. In eight kinds of human glioma cell lines, proliferation rate, expressions of metabolic enzymes and multidrug resistance genes, and drug efflux are tested. There were significant correlations in the expression levels of X gene and proliferation rate. The results indicate that MSCs can deliver suicide genes to intracranial tumors. These findings also highlight the potential advantages of autologous MSCs can provide a useful cellular vehicle to deliver suicide genes repetitively to suppress the recurrence of brain tumors. Also, the pre-diagnostic markers can make patients to be selected for the application of the therapy.
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TABLE OF CONTNENTS
ABSTRACT i
TABLE OF CONTENTS . iii
LIST OF FIGURES vi
LIST OF TABLES viii
I. INTRODUCTION 1
PART I 1
A. Mesenchymal stem cells 1
B. Tropism toward glioma 1
C. Glioma 2
D. Suicide gene therapy 2
PART II 5
A. 5-fluorouracil 5
B. 5-fluorouracil resistance mechanisms 6
1. Multidrug resistance genes 6
2. Metabolic enzymes 6
II. MATERIALS AND METHODS 9
PART I 9
A. Construction of retroviral vector expressing CD 9
B. Cell culture and transduction 9
C. In vitro suicide effects 10
D. In vitro bystander effects 11
E. Quantification of 5-FU by high performance liquid chromatography
(HPLC) 12
F. In vivo antitumor effects of MSC/CDs in SD-rat 12
G. In vivo antitumor effects of MSC/CDs in nude mouse 13
H. Statistics analysis 14
PART II 15
A. 5-FU sensitivity of glioma cells 15
B. Proliferation rates of glioma cell lines 15
C. Drug efflux of rhodamin 123 by fluorescence activated cell sorter
analysis 15
D. Drug efflux of radio-isotope labeled 5-fluorouracil 16
E. Reverse transcription polymerase chain reaction (RT-PCR) 16
III. RESULTS 20
PART I 20
A. CD-expressing mesenchymal stem cells by divergent LTRs 20
B. In vitro suicide effects 20
C. In vitro bystander effects 23
D. In vivo therapeutic effects 31
E. Requirement for multiple transplantations in pre-existing, established
brain tumors 36
F. Immunohistochemical analysis in the reduced tumor mass by CD expressing
MSCs 41
G. The multiple transplantation effects of MSC-FIP/CD in nude mouse 41
PART II 47
A. Diversity of glioma cell lines 47
B. Sensitivity to 5-FU 47
C. 5-FU resistance mechanisms 51
IV. DISCUSSION 57
V. CONCLUSION 62
REFERENCE 63
국문요약 71
