뇌종양 유래 페리오스틴의 사람 신경 줄기세포 유도
Glioma produced periostin attracts human neural stem cells
- 주제(키워드) Periostin , Neural stem cell , Tumor tropism , Bystander effect , Cancer therapy
- 발행기관 아주대학교
- 지도교수 이명애
- 발행년도 2010
- 학위수여년월 2010. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의학과
- 실제URI http://www.dcollection.net/handler/ajou/000000010865
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Stem cells from various lineages have been well known for their migration tendency toward glioma and become attractive vehicles to deliver therapeutic genes to brain tumors. However, which factors and mechanisms in these processes are not yet known. In the present study, I aimed at identifying the chemoattractant molecules which induce the tropism of human neural stem cell (hNSC) for brain tumor and characterizing their mode of mechanisms. For this, I performed microarray analyses of different type of brain tumors and identified 14 secretory proteins that were highly and specifically expressed in brain tumors. Real-time PCR assay and immunohistochemistry with various brain tumor samples showed that periostin (POSTN) among these factors, known for its role in angiogenesis and invasion during tumor development, was specifically expressed only in glioblastoma multiforms, malignancty tumors. We are also found that a recombinant human POSTN promoted migration of hNSCs in vitro with 3 times higher potency than VEGF. Transplantation of NIH3T3 cells in a rat brain expressing a secretable POSTN in one cerebral hemisphere induced migration of NSCs that were inoculated in the contralateral of hemisphere. We found that POSTN bound to integrin alphaV beta5 complex in NSCs promoted phosphorylation of focal adhesion kinase (FAK) and protein kinase B (AKT) through activation of the phosphorinositide 3-kinase (PI3K) pathway. Inhibition of CDK5 but not ERK suppress that signaling dramatically impaired the migration of hNSCs toward POSTN. NSC use similar signaling pathway during CNS development and glioma invasion Previous study demonstrated that hNSCs (F3.CD) that were retrovirally transduced with cytosine deaminase (CD) gene showed remarkable bystander effects on the glioma cells after application of the prodrug, 5-fluorocytosine (5-FC). To harness the chemotactic ability of POSTN as a tool to induce a strong bystander killer effect of brain tumors, we established the brain tumor animal model which was implanted with C6 cells and the increasing number of POSTN-overexpressing cells, and F3.CD cells were injected into the contralateral hemisphere followed by systemic 5-FC administration. Interestingly, increasing number of POSTN-overexpressing cells resulted in significant reduced tumor volumes, but not tumor cell numbers. While many tumor cells scattered around the original transplantation site in control tumor group, scattered cells became disappeared with increasing number of POSTN-releasing cells. This result demonstrated that the more POSTN was released, the faster hNSC migrated, which resulted in much NSCs at tumor site and more clearance of tumor cells. Taken together, POSTN treatment combined with bystander killer effect of NSCs might have strong therapeutic potential against aggressively invasive gliomas.
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TABLE OF CONTENTS
ABSTRACT ⅰ
TABLE OF CONTENTS ⅲ
LIST OF FITURES Ⅴ
LIST OF TABLES ⅶ
ABBREVIATION ⅷ
Ⅰ. INTRODUCTION 1
Ⅱ. MATERIAL AND METHODS 7
1. Cell culture 7
2. In vitro migration 7
3. Collection of normal brain and brain tumor tissue 8
4. RNA isolation and application to Affymetrix GeneChip 8
5. Real-time quantitative RT–PCR 9
6. Western Blotting Analysis 9
7. Reveres Transcription - PCR 10
8. Immunohistochemistry 11
9. Construction of Periostin overexpressing cell line 12
10. In vivo migration 13
11. Analysis of CD enzyme activity 14
12. In vitro bystander effect assay 14
12. In vivo bystander effect assay 15
13. Statistical analysis 15
Ⅲ. RESULTS 16
1. Expression profiling of secreted genes from human brain tumor tissues 16
2. Differential expression of 14 Candidate genes in several human brain tumor tissues 23
3. Periostin more intensely expressed in human brain tumor tissues than normal brain 26
4. Periostin strongly attracted neural stem cell than VEGF 29
5. Neural stem cell has motility to Periostin-expressing NIH3T3 in vivo condition 34
6. Peirostin Receptor expression in neural stem cell lines 42
7. FAK and CDK5 signal pathway was included in the neural stem cell migration 44
8. The neural stem cell migration regulated by DCX signal pathway 46
9. Cytotoxic effect of F3-CD cell to p-NIH3T3 cell 50
10. The therapeutic effect of F3-CD cells in rat glioma model 56
Ⅳ. DISCUSSION 62
Ⅴ. CONCLUSION 69
REFERENCES 70
국문요약 82
