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Characteristic analysis of mobility inducers for neural stem cells

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dc.contributor.author김, 정미-
dc.description.abstractRecently neural stem cells (NSCs) were reported to migrate to damaged brain areas, such as brain tumors or ischemic regions. Systemic strategy can be effective to cancer therapeutic effect through cell-based gene therapy. From our previous studies, we identified chemoattractant molecules-1(CM-1) and CM-2 as novel chemoattractants that induced tumor-tropism of neural stem cells, and showed that CM-1 accelerated tumor tropism of human NSCs encoding therapeutic gene and increased targeting in vivo model of rats. Previous our results showed that in rats transplanted with NIH 3T3 cells overexpressing CM-1 and glioma cells, the edge of tumor mass did not infiltrate and invasive tumor cells were eliminated from surrounding region compared with only glioma cells. So, we examined that CM-1 bound to the integrin αvβ5, known as candidate CM-1 receptor in NSCs and resulted in promoting activation of signal pathway for mobility. In migration assay using functional blocking antibody, we identified that integrin αvβ5 interacted with CM-1 and induced functional cell migration in human glioblastoma primary cultured cells and rat glioma cells as well as human NSCs. In the presence of CM-1, NSC migrated toward opposite area significantly compare with control group. According to that, CM-1 made brain cancer stem cells to assemble toward tumor mass. To investigate whether CM-1-induced NSC migration can have in vivo therapeutic efficacy, we determined survival time of brain tumor animal model transplanted with NSC harboring suicidal gene and CM-1. The result of the in vivo experiment for CM-1 showed that human NSCs encoding cytosine deaminase (CD) migrated towards co-transplantation sites of NIH3T3 cells overexpressing CM-1 and glioma cells compared to only glioma cells. In addition, bystander effect by repetitive infusion of prodrug, 5-fluorocytosine (5-FC), reduced tumor mass with increasing number of CM-2-expressing cells. Transplanted group of CM-1-producing NIH 3T3 cells significantly extended survival time over 105 days than transplantation of glioma cells. PET imaging data showed that increase of survival correlated with reduction of tumor mass volume. Our previous study showed that, the other Chemoattractants molecule, CM-2 induced NSCs migration by interacting with CD63 and CM-2-CD63complex bound to integrin β1. To investigate the detailed molecular mechanism for NSCs migration by CM-2, in migration assay, we confirmed that integrin β1 and PI3K signaling pathways were implicated in the induction of NSCs migration via CM-2. We investigated phosphorylation of tyrosine 397 site of FAK by using CD63-downregulated HB1.F3 cells with shRNA strategy and pREP4 F3 cells. Our results showed that human NSCs activated phosphorylation of FAK by CM-2 for migration with the exception of shCD63 knockout NSCs. These findings provide us an important step towards the development of new NSCs based gene therapies to cure brain tumor and hold promise for use as a therapeutic approach to treat aggressively invasive gliomas such as glioblastoma multiforme.-
dc.description.abstract종양-굴성을 가진 신경줄기세포는 종양을 표적화 하는 치료적 방법에 용이 할 수 있다. 이러한 종양-굴성을 향상시킬 수 있는 CM-1과 CM-2는 이전에 인간 뇌조직의 microarray를 통해 과-발현되는 것이 확인 되었다. 이전 연구는 여러 환자의 뇌종양에서 이동성 유도물질로서 CM-1와 CM-2가 일반적으로 높게 발현되며, 이 물질들이 신경줄기세포의 이동성을 상당히 향상시킴을 확인했다. 또한 이전의 결과는 우리는 F3 신경줄기세포뿐만 아니라 C6 쥐 신경교종 세포와 인간의 신경교종 조직에서 배양된 세포에 존재하는 CM-1의 수용체로 인테그린 αvβ5을 확인했다. 이에 따라 뇌 암줄기세포들도 CM-1가 높은 농도로 존재하는, 종양 덩어리 쪽으로 모이게 만들었다. 이에 따라 우리는 In vivo, 쥐의 뇌종양 모델을 설립하였고, CM-1에 의해 유도되어 이동한 F3.CD에서 분비된 cytosine deaminase는 전구약물인 5-fluorocytosine와의 작용을 통해 종양의 크기가 줄어듦을 확인하였다. 또한 이 실험을 통해 방관자 효과 및 신경줄기세포 이동성 향상이 생존 시간의 연장에 상당히 효과적임을 밝혔다. 다른 이동성 유도물질인 CM-2는 CD63 수용체에 의해 세포 이동을 유도하는 것으로 알려져 있다. CM-2에 의한 신경줄기세포의 이동성 유도에 있어서 분자적 메커니즘을 조사하기 위해 migration assay를 수행하였고, 이를 통해 인테그린 β1, PI3K 신호 경로가 CM-2에 의한 신경 줄기 세포 이동의 유도와 연루되어 있다는 것을 확인했다. 또한, CD63 knockouted F3 세포와 pREP4 F3 세포에서 FAK의 타이로신 397자리에 인산화를 확인해본 결과, CD63이 발현되지 않는 신경줄기세포에서 CM-2에 의한 FAK 인산화는 pREP4 F3 세포에 비해 상당히 감소하였다. 종합적으로, 본 내용은 이동성 유도 물질들에 의해 종양-표적화 능력이 향상된 인간 신경 줄기 세포가 독창적인 암 치료법으로 이용될 수 있음을 시사한다.-
dc.description.tableofcontentsABSTRACT ⅰ





A. Cells and cell culture 6

B. Brain tumor animal model 6

C. 5-FC treatment 7

D. PET scan and tumor volume evaluation 7

E. Blocking antibody migration assay 8

F. Boydenchamber assay 9

G. Western blot analysis 9


A. Cancer stem cells induced migration by CM-1 via integrin αvβ5 as receptor of CM-1 as well as NSCs 11

B. Systemic strategy using CM-1 as chemoattractants and CD-5-FC was effective to prolong survival time 15

C. CM-2 was crucial interaction with integrin β1 in induction of NSCs migration 21

D. FAK and PI3K signal pathway were involved in the NSCs migration 23




국문요약 35
dc.titleCharacteristic analysis of mobility inducers for neural stem cells-
dc.title.alternative신경줄기세포에 대한 이동성 유도물질들의 특성 분석-
dc.subject.keywordNeural stem cell-
dc.subject.keywordTumor tropism-
dc.subject.keywordStem cell therapy-
dc.subject.keywordGlioblastoma multiforme (GBM)-
dc.subject.keywordMobility inducers for neural stem cells-
dc.contributor.department대학원 의생명과학과-
dc.contributor.affiliatedAuthor김, 정미-
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